FIU Biochemistry Textbook Chapter Summaries (Final Study Guide)
FIU Biochemistry Textbook Chapter Summaries (Final Study Guide) BCH 3033
Popular in FIU Biochemistry
Popular in Biology
This 21 page Study Guide was uploaded by Pelin Darendeliler on Wednesday December 17, 2014. The Study Guide belongs to BCH 3033 at University of Miami taught by John Makemson in Spring2014. Since its upload, it has received 416 views. For similar materials see FIU Biochemistry in Biology at University of Miami.
Reviews for FIU Biochemistry Textbook Chapter Summaries (Final Study Guide)
Lectures notes?? Yes please! Looking forward to the next set!
-Mr. Bridgette Kuvalis
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 12/17/14
SUMMARY 11 Cellular Foundations All cells are bounded by a plasma membrane have a cytosol containing metabolites coenzymes inorganic ions and enzymes and have a set of genes contained within a nucleoid prokaryotes or nucleus eukaryotes Phototrophs use sunlight to do work chemotrophs oxidize fuels passing electrons to good electron acceptors inorganic compounds organic compounds or molecular oxygen Bacterial cells contain cytosol a nucleoid and plasmids Eukaryotic cells have a nucleus and are multicompartmented segregating certain processes in specific organelles which can be separated and studied in isolation Cytoskeletal proteins assemble into long filaments that give cells shape and rigidity and serve as rails along which cellular organelles move throughout the cell 1 Supramolecular complexes are held together by noncovalent interactions and form a hierarchy of structures some visible with the light microscope When individual molecules are removed from these complexes to be studied in vitro interactions important in the living cell may be lost SUMMARY 12 Chemical Foundations I Because of its bonding versatility carbon can produce a broad array of carbon carbon skeletons with a variety of functional groups these groups give biomolecules their biological and chemical personalities I A nearly universal set of several hundred small molecules is found in living cells the interconver sions of these molecules in the central metabolic pathways have been conserved in evolution I Proteins and nucleic acids are linear polymers of simple monomeric subunits their sequences contain the information that gives each molecule its threedimensional structure and its biological functions Molecular configuration can be changed only by breaking covalent bonds For a carbon atom with four different substituents a chiral carbon the substituent groups can be arranged in two different ways generating stereoisomers with distinct properties Only one stereoisomer is biologically active Molecular conformation is the position of atoms in space that can be changed by rotation about single bonds without breaking covalent bonds Interactions between biological molecules are almost invariably stereospecific they require a complementary match between the interacting molecules SUMMARY 13 Physical Foundations I Living cells are open systems exchanging matter and energy with their surroundings extracting and channeling energy to maintain themselves in a dynamic steady state distant from equilibrium Energy is obtained from sunlight or fuels by converting the energy from electron ow into the chemical bonds of ATP I The tendency for a chemical reaction to proceed toward equilibrium can be expressed as the freeenergy change G which has two components enthalpy change H and entropy change S These variables are related by the equation G H T S I When G of a reaction is negative the reaction is exergonic and tends to go toward completion when G is positive the reaction is endergonic and tends to go in the reverse direction When two reactions can be summed to yield a third reaction the G for this overall reaction is the sum of the Gs of the two separate reactions This provides a way to couple reactions I The conversion of ATP to Pi and ADP is highly exergonic large negative G and many endergonic cellular reactions are driven by coupling them through a common intermediate to this reaction I The standard freeenergy change for a reaction G is a physical constant that is related to the equilibrium constant by the equation G RT ln Keq I Most exergonic cellular reactions proceed at useful rates only because enzymes are present to catalyze them Enzymes act in part by stabilizing the transition state reducing the activation energy GI and increasing the reaction rate by many orders of magnitude The catalytic activity of enzymes in cells is regulated I Metabolism is the sum of many interconnected reaction sequences that interconvert cellular metabolites Each sequence is regulated so as to provide what the cell needs at a given time and to expend energy only when necessary SUMMARY 14 Genetic Foundations Genetic information is encoded in the linear sequence of four deoxyribonucleotides in DNA The doublehelical DNA molecule contains an internal template for its own replication and repair The linear sequence of amino acids in a pro tein which is encoded in the DNA of the gene for that protein produces a protein s unique threedimensional structure Individual macromolecules with specific affinity for other macromolecules selfassemble into supramolecular complexes SUMMARY 15 Evolutionary Foundations Occasional inheritable mutations yield an organism that is better suited for survival in an ecological niche and progeny that are preferentially selected This process of mutation and selection is the basis for the Darwinian evolution that led from the first cell to all the organisms that now exist and it explains the fundamental similarity of all living organisms Life originated about 35 billion years ago most likely with the formation of a membraneenclosed compartment containing a selfreplicating RNA molecule The components for the rst cell were produced by the action of lightning and high temperature on simple atmospheric molecules such as CO2 and NH3 The catalytic and genetic roles of the early RNA genome were separated over time with DNA becoming the genomic material and proteins the major catalytic species Eukaryotic cells acquired the capacity for photosynthesis and for oxidative phosphorylation from endosymbiotic bacteria In multicellular organisms differentiated cell types specialize in one or more of the functions essential to the organism s survival Knowledge of the complete genomic nucleotide sequences of organisms from different branches of the phylogenetic tree provides insights into the evolution and function of extant organisms and offers great opportunities in human medicine SUMMARY 21 Weak Interactions in Aqueous Systems I The very different electronegativities of H and 0 make water a highly polar molecule capable of forming hydrogen bonds with itself and with solutes Hydrogen bonds are eeting primarily electrostatic and weaker than covalent bonds Water is a good solvent for polar hydrophilic solutes with which it forms hydrogen bonds and for charged solutes with which it interacts electrostatically I Nonpolar hydrophobic compounds dissolve poorly in water they cannot hydrogenbond with the solvent and their presence forces an energetically unfavorable ordering of water molecules at their hydrophobic surfaces To minimize the surface exposed to water nonpolar compounds such as lipids form aggregates micelles in which the hydrophobic moieties are sequestered in the interior associating through hydrophobic interactions and only the more polar moieties interact with water I Numerous weak noncovalent interactions deci sively in uence the folding of macromolecules such as proteins and nucleic acids The most stable macromolecular conformations are those in which hydrogen bonding is maximized within the molecule and between the molecule and the solvent and in which hydrophobic moieties cluster in the interior of the molecule away from the aqueous solvent I The physical properties of aqueous solutions are strongly in uenced by the concentrations of solutes When two aqueous compartments are separated by a semipermeable membrane such as the plasma membrane separating a cell from its surroundings water moves across that membrane to equalize the osmolarity in the two compartments This tendency for water to move across a semipermeable membrane is the osmotic pressure SUMMARY 22 Ionization of Water Weak Acids and Weak Bases I Pure water ionizes slightly forming equal num bers of hydrogen ions hydronium ions H30 and hydroxide ions The extent of ionization is described by an equilibrium constant Keq H OH h20 from which the ion product of H20 water KW is derived At25 C KW H OH 555 MKeq10 14 M2 I The pH of an aqueous solution re ects on a logarithmic scale the concentration of 1 hydrogen ions pH log log H I The greater the acidity of a solution the lower its pH Weak acids partially ionize to release a hydrogen ion thus lowering the pH of the aqueous solution Weak bases accept a hydro gen ion increasing the pH The extent of these processes is characteristic of each particular weak acid or base and is expressed as a dissociation constant Ka Keq Ka HA I The pKa expresses on a logarithmic scale the relative strength of a weak acid or basel p K a l o g l o g K a The stronger the acid the lower its pKa the stronger the base the higher its pKa The pKa can be determined experimentally it is the pH at the midpoint of the titration curve for the acid or base SUMMARY 23 Buffering against pH Changes in Biological Systems I A mixture of a weak acid or base and its salt resists changes in pH caused by the addition of H or OH The mixture thus functions as a buffer I The pH of a solution of a weak acid or base and its salt is given by the HendersonHA Hasselbalch equation In cells and tissues phosphate and bicarbonate buffer systems maintain intracellular and extra cellular uids at their optimum physiological pH which is usually close to pH 7 Enzymes generally work optimally at this pH SUMMARY 24 Water as a Reactant I Water is both the solvent in which metabolic reactions occur and a reactant in many bio chemical processes including hydrolysis con densation and oxidationreduction reactions SUMMARY 31 Amino Acids The 20 amino acids commonly found as residues in proteins contain an carboxyl group an amino group and a distinctive R group substituted on the carbon atom The carbon atom of all amino acids except glycine is asymmetric and thus amino acids can exist in at least two stereoisomeric forms Only the L stereoisomers with a con guration related to the absolute con guration of the reference molecule Lglyceraldehyde are found in proteins Other less common amino acids also occur either as constituents of proteins through modification of common amino acid residues after protein synthesis or as free metabolites Amino acids are classified into ve types on the basis of the polarity and charge at pH 7 of their R groups Amino acids vary in their acidbase properties and have characteristic titration curves Monoamino monocarboxylic amino acids with nonionizable R groups are diprotic acids H3NCHRCOOH at low pH and exist in several different ionic forms as the pH is increased Amino acids with ionizable R groups have additional ionic species depending on the pH of the medium and the pKa of the R group SUMMARY 32 Peptides and Proteins I Amino acids can be joined covalently through peptide bonds to form peptides and proteins Cells generally contain thousands of different proteins each with a different biological activity I Proteins can be very long polypeptide chains of 100 to several thousand amino acid residues However some naturally occurring peptides have only a few amino acid residues Some proteins are composed of several noncovalently associated polypeptide chains called subunits Simple proteins yield only amino acids on hydrolysis conjugated proteins contain in addition some other component such as a metal or organic prosthetic group I The sequence of amino acids in a protein is characteristic of that protein and is called its primary structure This is one of four generally recognized levels of protein structure SUMMARY 33 Working with Proteins Proteins are separated and purified by taking advantage of differences in their properties Proteins can be selectively precipitated by the addition of certain salts A wide range of chromatographic procedures makes use of differences in size binding affinities charge and other properties These include ion exchange sizeexclusion affinity and high performance liquid chromatography Electrophoresis separates proteins on the basis of mass or charge SDS gel electrophoresis and isoelectric focusing can be used separately or in combination for higher resolution All purification procedures require a method for quantifying or assaying the protein of interest in the presence of other proteins Purification can be monitored by assaying specific activity SUMMARY 34 The Covalent Structure of Proteins Differences in protein function result from differences in amino acid composition and sequence Some variations in sequence are possible for a particular protein with little or no effect on function Amino acid sequences are deduced by fragmenting polypeptides into smaller peptides using reagents known to cleave specific peptide bonds determining the amino acid sequence of each fragment by the automated Edman degradation procedure then ordering the peptide fragments by finding sequence overlaps between fragments generated by different reagents A protein sequence can also be deduced from the nucleotide sequence of its corresponding gene in DNA Short proteins and peptides up to about 100 residues can be chemically synthesized The peptide is built up one amino acid residue at a time while remaining tethered to a solid support SUMMARY 35 Protein Sequences and Evolution I Protein sequences are a rich source of information about protein structure and function as well as the evolution of life on this planet Sophisticated methods are being developed to trace evolution by analyzing the resultant slow changes in the amino acid sequences of homologous proteins SUMMARY 41 Overview of Protein Structure Every protein has a threedimensional structure that re ects its function Protein structure is stabilized by multiple weak interactions Hydrophobic interactions are the major contributors to stabilizing the globular form of most soluble proteins hydrogen bonds and ionic interactions are optimized in the specific structures that are thermodynamically most stable The nature of the covalent bonds in the polypeptide backbone places constraints on structure The peptide bond has a partial double bond character that keeps the entire sixatom peptide group in a rigid planar configuration The NOC and C OC bonds can rotate to assume bond angles of and respectively SUMMARY 42 Protein Secondary Structure I Secondary structure is the regular arrangement of amino acid residues in a segment of a polypeptide chain in which each residue is spatially related to its neighbors in the same way I The most common secondary structures are the helix the conformation and turns I The secondary structure of a polypeptide segment can be completely defined if the and angles are known for all amino acid residues in that segment SUMMARY 43 Protein Tertiary and Quaternary Structures I Tertiary structure is the complete three dimensional structure of a polypeptide chain There are two general classes of proteins based on tertiary structure fibrous and globular I Fibrous proteins which serve mainly structural roles have simple repeating elements of secondary structure I Globular proteins have more complicated tertiary structures often containing several types of secondary structure in the same polypeptide chain The first globular protein structure to be determined using xray diffraction methods was that of myoglobin I The complex structures of globular proteins can be analyzed by examining stable substructures called supersecondary structures motifs or folds The thousands of known protein structures are generally assembled from a repertoire of only a few hundred motifs Regions of a polypeptide chain that can fold stably and independently are called domains I Quaternary structure results from interactions between the subunits of multisubunit multimeric proteins or large protein assemblies Some multimeric proteins have a repeated unit consisting of a single subunit or a group of subunits referred to as a protomer Protomers are usually related by rotational or helical symmetry SUMMARY 44 Protein Denaturation and Folding I The threedimensional structure and the function of proteins can be destroyed by denaturation demonstrating a relationship between structure and function Some denatured proteins can renature spontaneously to form biologically active protein showing that protein tertiary structure is determined by amino acid sequence I Protein folding in cells probably involves multiple pathways Initially regions of secondary structure may form followed by folding into supersecondary structures Large ensembles of folding intermediates are rapidly brought to a single native conformation I For many proteins folding is facilitated by Hsp70 chaperones and by chaperonins Disulfide bond formation and the cis trans isomerization of Pro peptide bonds are catalyzed by specific enzymes SUMMARY 51 Reversible Binding of a Protein to a Ligand OxygenBinding Proteins Protein function often entails interactions with other molecules A molecule bound by a protein is called a ligand and the site to which it binds is called the binding site Proteins may undergo conformational changes when a ligand binds a process called induced fit In a multisubunit protein the binding of a ligand to one subunit may affect ligand binding to other subunits Ligand binding can be regulated Myoglobin contains a heme prosthetic group which binds oxygen Heme consists of a single atom of Fez coordinated within a porphyrin Oxygen binds to myoglobin reversibly this simple reversible binding can be described by an association constant Ka or a dissociation constant Kd For a monomeric protein such as myoglobin the fraction of binding sites occupied by a ligand is a hyperbolic function of ligand concentration I Normal adult hemoglobin has four heme containing subunits two and two similar in structure to each other and to myoglobin Hemoglobin exists in two interchangeable structural states T and R The T state is most stable when oxygen is not bound Oxygen binding promotes transition to the R state I Oxygen binding to hemoglobin is both allosteric and cooperative As O2 binds to one binding site the hemoglobin undergoes conformational changes that affect the other binding sites an example of allosteric behavior Conformational changes between the T and R states mediated by subunitsubunit interactions result in cooperative binding this is described by a sigmoid binding curve and can be analyzed by a Hill plot I Two major models have been proposed to explain the cooperative binding of ligands to multisubunit proteins the concerted model and the sequential model I Hemoglobin also binds H and CO2 resulting in the formation of ion pairs that stabilize the T state and lessen the protein s affinity for O2 the Bohr effect Oxygen binding to hemoglobin is also modulated by 23 bisphosphoglycerate which binds to and stabilizes the T state I Sicklecell anemia is a genetic disease caused by a single amino acid substitution Glu6 to Val6 in each chain of hemoglobin The change produces a hydrophobic patch on the surface of the hemoglobin that causes the molecules to aggregate into bundles of fibers This homozygous condition results in serious medical complications SUMMARY 52 Complementary Interactions between Proteins and Ligands The Immune System and Immunoglobulins The immune response is mediated by interactions among an array of specialized leukocytes and their associated proteins T lymphocytes produce Tcell receptors B lymphocytes produce immunoglobulins All cells produce MHC proteins which display host self or antigenic nonself peptides on the cell surface In a process called clonal selection helper T cells induce the proliferation of B cells and cytotoxic T cells that produce immunoglobulins or of Tcell receptors that bind to a specific antigen Humans have five classes of immunoglobulins each with different biological functions The most abundant class is IgG a Y shaped protein with two heavy and two light chains The domains near the upper ends of the Y are hypervariable within the broad population of IgGs and form two antigenbinding sites A given immunoglobulin generally binds to only a part called the epitope of a large antigen Binding often involves a conformational change in the IgG an induced fit to the antigen SUMMARY 53 Protein Interactions Modulated by Chemical Energy Actin Myosin and Molecular Motors I Proteinligand interactions achieve a special degree of spatial and temporal organization in motor proteins Muscle contraction results from choreographed interactions between myosin and actin coupled to the hydrolysis of ATP by myosin I Myosin consists of two heavy and four light chains forming a fibrous coiled coil tail domain and a globular head domain Myosin molecules are organized into thick filaments which slide past thin filaments composed largely of actin ATP hydrolysis in myosin is coupled to a series of conformational changes in the myosin head leading to dissociation of myosin from one Factin subunit and its eventual reassociation with another farther along the thin filament The myosin thus slides along the actin filaments I Muscle contraction is stimulated by the release of Ca2 from the sarcoplasmic reticulum The Ca2 binds to the protein troponin leading to a conformational change in a troponintropomyosin complex that triggers the cycle of actinmyosin interactions SUMMARY 61 An Introduction to Enzymes Life depends on the existence of powerful and specific catalysts the enzymes Almost every biochemical reaction is catalyzed by an enzyme With the exception of a few catalytic RNAs all known enzymes are proteins Many require nonprotein coenzymes or cofactors for their catalytic function Enzymes are classified according to the type of reaction they catalyze All enzymes have formal EC numbers and names and most have trivial names SUMMARY 62 How Enzymes Work 17 Enzymes are highly effective catalysts commonly enhancing reaction rates by a factor of 105 to 10 Enzymecatalyzed reactions are characterized by the formation of a complex between substrate and enzyme an ES complex Substrate binding occurs in a pocket on the enzyme called the active site The function of enzymes and other catalysts is to lower the activation energy GI for a reaction and thereby enhance the reaction rate The equilibrium of a reaction is unaffected by the enzyme A significant part of the energy used for enzymatic rate enhancements is derived from weak interactions hydrogen bonds and hydrophobic and ionic interactions between substrate and enzyme The enzyme active site is structured so that some of these weak interactions occur preferentially in the reaction transition state thus stabilizing the transition state The need for multiple interactions is one reason for the large size of enzymes The binding energy GB can be used to lower substrate entropy or to cause a conformational change in the enzyme induced fit Binding energy also accounts for the exquisite specificity of enzymes for their substrates I Additional catalytic mechanisms employed by enzymes include general acidbase catalysis covalent catalysis and metal ion catalysis Catalysis often involves transient covalent interactions between the substrate and the enzyme or group transfers to and from the enzyme so as to provide a new lowerenergy reaction path SUMMARY 63 Enzyme Kinetics As an Approach to Understanding Mechanism I Most enzymes have certain kinetic properties in common When substrate is added to an enzyme the reaction rapidly achieves a steady state in which the rate at which the ES complex forms balances the rate at which it reacts As S increases the steadystate activity of a fixed concentration of enzyme increases in a hyperbolic fashion to approach a characteristic maximum rate Vmax at which essentially all the enzyme has formed a complex with substrate I The substrate concentration that results in a reaction rate equal to onehalf Vmax is the Michaelis constant Km which is characteristic for each enzyme acting on a given substrate The MichaelisMenten equation Vo Vmax S Km S relates initial velocity to S and Vmax through the constant Km MichaelisMenten kinetics is also called steadystate kinetics I Km and Vmax have different meanings for different enzymes The limiting rate of an enzymecatalyzed reaction at saturation is described by the constant kcat the turnover number The ratio kcatKm provides a good measure of catalytic efficiency The Michaelis Menten equation is also applicable to bisubstrate reactions which occur by ternarycomplex or PingPong doubledisplacement pathways Reversible inhibition of an enzyme is competitive uncompetitive or mixed Competitive inhibitors compete with substrate by binding reversibly to the active site but they are not transformed by the enzyme Uncompetitive inhibitors bind only to the ES complex at a site distinct from the active site Mixed inhibitors bind to either E or ES again at a site distinct from the active site In irreversible inhibition an inhibitor binds permanently to an active site by forming a covalent bond or a very stable noncovalent interaction Every enzyme has an optimum pH or pH range at which it has maximal activity SUMMARY 64 Examples of Enzymatic Reactions Chymotrypsin is a serine protease with a well understood mechanism featuring general acid base catalysis covalent catalysis and transitionstate stabilization Hexokinase provides an excellent example of induced fit as a means of using substrate binding energy The enolase reaction proceeds via metal ion catalysis Lysozyme makes use of covalent catalysis and general acid catalysis as it promotes two successive nucleophilic displacement reactions SUMMARY 65 Regulatory Enzymes I The activities of metabolic pathways in cells are regulated by control of the activities of certain enzymes I In feedback inhibition the end product of a pathway inhibits the first enzyme of that pathway I The activity of allosteric enzymes is adjusted by reversible binding of a specific modulator to a regulatory site Modulators may be the substrate itself or some other metabolite and the effect of the modulator may be inhibitory or stimulatory The kinetic behavior of allosteric enzymes re ects cooperative interactions among enzyme subunits Other regulatory enzymes are modulated by covalent modification of a specific functional group necessary for activity The phosphorylation of specific amino acid residues is a particularly common way to regulate enzyme activity Many proteolytic enzymes are synthesized as inactive precursors called zymogens which are activated by cleavage of small peptide fragments Enzymes at important metabolic intersections may be regulated by complex combinations of effectors allowing coordination of the activities of interconnected pathways SUMMARY 71 Monosaccharides and Disaccharides Sugars also called saccharides are compounds containing an aldehyde or ketone group and two or more hydroxyl groups Monosaccharides generally contain several chiral carbons and therefore exist in a variety of stereochemical forms which may be represented on paper as Fischer projections Epimers are sugars that differ in configuration at only one carbon atom Monosaccharides commonly form internal hemiacetals or hemiketals in which the aldehyde or ketone group joins with a hydroxyl group of the same molecule creating a cyclic structure this can be represented as a Haworth perspective formula The carbon atom originally found in the aldehyde or ketone group the anomeric carbon can assume either of two configurations and which are interconvertible by mutarotation In the linear form which is in equilibrium with the cyclized forms the anomeric carbon is easily oxidized A hydroxyl group of one monosaccharide can add to the anomeric carbon of a second monosaccharide to form an acetal In this disaccharide the glycosidic bond protects the anomeric carbon from oxidation Oligosaccharides are short polymers of several monosaccharides joined by glycosidic bonds At one end of the chain the reducing end is a monosaccharide unit whose anomeric carbon is not involved in a glycosidic bond The common nomenclature for di or oligosaccharides specifies the order of monosaccharide units the configuration at each anomeric carbon and the carbon atoms involved in the glycosidic linkages SUMMARY 72 Polysaccharides I Polysaccharides glycans serve as stored fuel and as structural components of cell walls and extracellular matrix I The homopolysaccharides starch and glycogen are stored fuels in plant animal and bacterial cells They consist of D glucose with linkages and all three contain some branches I The homopolysaccharides cellulose chitin and dextran serve structural roles Cellulose composed of ln4linked D glucose residues lends strength and rigidity to plant cell walls Chitin a polymer of ln4linked Nacetylglucosamine strengthens the exoskeletons of arthropods Dextran forms an adhesive coat around certain bacteria Homopolysaccharides fold in three dimensions The chair form of the pyranose ring is essentially rigid so the conformation of the polymers is determined by rotation about the bonds to the oxygen on the anomeric carbon Starch and glycogen form helical structures with intrachain hydrogen bonding cellulose and chitin form long straight strands that interact with neighboring strands Bacterial and algal cell walls are strengthened by heteropolysaccharides peptidoglycan in bacteria agar in red algae The repeating disaccharide in peptidoglycan is GlcNAc ln4Mur2Ac in agarose it is DGal ln436anhydroLGal Glycosaminoglycans are extracellular hetero polysaccharides in which one of the two monosaccharide units is a uronic acid and the other an Nacetylated amino sugar Sulfate esters on some of the hydroxyl groups give these polymers a high density of negative charge forcing them to assume extended conformations These polymers hyaluronate chondroitin sulfate dermatan sulfate keratan sulfate and heparin provide viscosity adhesiveness and tensile strength to the extracellular matrix SUMMARY 73 Glycoconjugates Proteoglycans Glycoproteins and Glycolipids Proteoglycans are glycoconjugates in which a core protein is attached covalently to one or more large glycans such as heparan sulfate chondroitin sulfate or keratan sulfate The glycan is the greater portion by mass of the molecule Bound to the outside of the plasma membrane by a transmembrane peptide or a covalently attached lipid proteoglycans provide points of adhesion recognition and information transfer between cells or between the cell and the extracellular matrix Glycoproteins contain covalently linked oligosaccharides that are smaller but more structurally complex and therefore more informationrich than glycosaminoglycans Many cell surface or extracellular proteins are glycoproteins as are most secreted proteins The covalently attached oligosaccharides in uence the folding and stability of the proteins provide critical information about the targeting of newly synthesized proteins and allow for specific recognition by other proteins I Glycolipids and lipopolysaccharides are components of the plasma membrane with covalently attached oligosaccharide chains exposed on the cell s outer surface SUMMARY 74 Carbohydrates as Informational Molecules The Sugar Code Monosaccharides can be assembled into an almost limitless variety of oligosaccharides which differ in the stereochemistry and position of glycosidic bonds the type and orientation of substituent groups and the number and type I of branches Oligosaccharides are far more informationdense than nucleic acids or proteins Lectins proteins with highly specific carbohydratebinding domains are commonly found on the outer surface of cells where they initiate interaction with other cells In vertebrates oligosaccharide tags read by lectins govern the rate of degradation of certain peptide hormones circulating proteins and blood cells The adhesion of bacterial and viral pathogens to their animalcell targets occurs through binding of lectins in the pathogens to oligosaccharides in the target cell surface Lectins are also present inside cells where they mediate intracellular protein targeting Xray crystallography of lectinsugar complexes shows the detailed complementarity between the two molecules which accounts for the strength and specificity of their interactions with carbohydrates Selectins are plasma membrane lectins that bind carbohydrate chains in the extracellular matrix or on the surfaces of other cells thereby mediating the ow of information between cell and matrix or between cells SUMMARY 75 Working with Carbohydrates I Establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions the sequence in each branch the configuration of each monosaccharide unit and the positions of the glycosidic links a more complex problem than protein and nucleic acid analysis I The structures of oligosaccharides and polysaccharides are usually determined by a combination of methods specific enzymatic hydrolysis to determine stereochemistry and produce smaller fragments for further analysis methylation analysis to locate glycosidic bonds and stepwise degradation to determine sequence and configuration of anomeric carbons I Mass spectrometry and highresolution NMR spectroscopy applicable to small samples of carbohydrate yield essential information about sequence configuration at anomeric and other carbons and positions of glycosidic bonds SUMMARY 81 Some Basics A nucleotide consists of a nitrogenous base purine or pyrimidine a pentose sugar and one or more phosphate groups Nucleic acids are polymers of nucleotides joined together by phosphodiester linkages between the 5 hydroxyl group of one pentose and the 3 hydroxyl group of the next There are two types of nucleic acid RNA and DNA The nucleotides in RNA contain ribose and the common pyrimidine bases are uracil and cytosine In DNA the nucleotides contain 2 deoxyribose and the common pyrimidine bases are thymine and cytosine The primary purines are adenine and guanine in both RNA and DNA SUMMARY 82 Nucleic Acid Structure I Many lines of evidence show that DNA bears genetic information In particular the Avery MacLeodMcCarty experiment showed that DNA isolated from one bacterial strain can enter and transform the cells of another strain endowing it with some of the inheritable characteristics of the donor The HersheyChase experiment showed that the DNA of a bacterial virus but not its protein coat carries the genetic message for replication of the virus in a host cell I Putting together much published data Watson and Crick postulated that native DNA consists of two antiparallel chains in a righthanded doublehelical arrangement Complementary base pairs AUT and GqC are formed by hydrogen bonding within the helix The base pairs are stacked perpendicular to the long axis of the double helix 34 A apart with 105 base pairs per turn DNA can exist in several structural forms Two variations of the WatsonCrick form or BDNA are A and ZDNA Some sequencedependent structural variations cause bends in the DNA molecule DNA strands with appropriate se quences can form hairpincruciform structures or triplex or tetraplex DNA Messenger RNA transfers genetic information from DNA to ribosomes for protein synthesis Transfer RNA and ribosomal RNA are also involved in protein synthesis RNA can be structurally complex single RNA strands can be folded into hairpins doublestranded re gions or complex loops SUMMARY 83 Nucleic Acid Chemistry Native DNA undergoes reversible unwinding and separation of strands melting on heating or at extremes of pH DNAs rich in GqC pairs have higher melting points than DNAs rich in AUT pairs Denatured singlestranded DNAs from two species can form a hybrid duplex the degree of hybridization depending on the extent of sequence similarity Hybridization is the basis for important techniques used to study and isolate specific genes and RNAs DNA is a relatively stable polymer Spontaneous reactions such as deamination of certain bases hydrolysis of basesugar N glycosyl bonds radiationinduced formation of pyrimidine dimers and oxidative damage occur at very low rates yet are important because of cells very low tolerance for changes in genetic material DNA sequences can be determined and DNA polymers synthesized with simple automated protocols involving chemical and enzymatic methods SUMMARY 84 Other Functions of Nucleotides ATP is the central carrier of chemical energy in cells The presence of an adenosine moiety in a variety of enzyme cofactors may be related to bindingenergy requirements Cyclic AMP formed from ATP in a reaction catalyzed by adenylyl cyclase is a common second messenger produced in response to hormones and other chemical signals SUMMARY 91 DNA Cloning The Basics DNA cloning and genetic engineering involve the cleavage of DNA and assembly of DNA segments in new combinations recombinant DNA Cloning entails cutting DNA into fragments with enzymes selecting and possibly modifying a fragment of interest inserting the DNA fragment into a suitable cloning vector transferring the vector with the DNA insert into a host cell for replication and identifying and selecting cells that contain the DNA fragment Key enzymes in gene cloning include restriction endonucleases especially the type II enzymes and DNA ligase Cloning vectors include plasmids bacteriophages and for the longest DNA inserts bacterial artificial chromosomes BACs and yeast artificial chromosomes YACs Cells containing particular DNA sequences can be identified by DNA hybridization methods Genetic engineering techniques manipulate cells to express andor alter cloned genes SUMMARY 92 From Genes to Genomes The science of genomics broadly encompasses the study of genomes and their gene content Genomic DNA segments can be organized in libraries such as genomic libraries and cDNA libraries with a wide range of designs and purposes The polymerase chain reaction PCR can be used to amplify selected DNA segments from a DNA library or an entire genome In an international cooperative research effort the genomes of many organisms including that of humans have been sequenced in their entirety and are now available in public databases SUMMARY 93 From Genomes to Proteomes A proteome is the complement of proteins produced by a cell s genome The new field of proteomics encompasses an effort to catalog and determine the functions of all the proteins in a cell One of the most effective ways to determine the function of a new gene is by comparative genomics the search of databases for genes with similar sequences Paralogs and orthologs are proteins and their genes with clear functional and sequence relationships in the same or in different species In some cases the presence of a gene in combination with certain other genes observed as a pattern in several genomes can point toward a possible function Cellular proteomes can be displayed by two dimensional gel electrophoresis and explored with the aid of mass spectrometry The cellular function of a protein can sometimes be inferred by determining when and where its gene is expressed Researchers use DNA microarrays chips and protein chips to explore gene expression at the cellular level I Several new techniques including comparative genomics immunoprecipitation and yeast two hybrid analysis can identify proteinprotein interactions These interactions provide important clues to protein function SUMMARY 94 Genome Alterations and New Products of Biotechnology I Advances in whole genome sequencing and genetic engineering methods are enhancing our ability to modify genomes in all species Cloning in plants which makes use of the Ti plasmid vector from Agrobacterium allows the introduction of new plant traits In animal cloning researchers introduce foreign DNA primarily with the use of viral vectors or microinjection These techniques can produce transgenic animals and provide new methods for human gene therapy The use of genomics and proteomics in basic and pharmaceutical research is greatly advancing the discovery of new drugs Biotechnology is also generating an everexpanding range of other products and technologies SUMMARY 101 Storage Lipids I Lipids are waterinsoluble cellular components of diverse structure that can be extracted by nonpolar solvents I Almost all fatty acids the hydrocarbon components of many lipids have an even number of carbon atoms usually 12 to 24 they are either saturated or unsaturated with double bonds almost always in the cis configuration I Triacylglycerols contain three fatty acid molecules esterified to the three hydroxyl groups of glycerol Simple triacylglycerols contain only one type of fatty acid mixed triacylglycerols two or three types Triacylglycerols are primarily storage fats they are present in many foods SUMMARY 102 Structural Lipids in Membranes I The polar lipids with polar heads and nonpolar tails are major components of membranes The most abundant are the glycerophospholipids which contain fatty acids esterified to two of the hydroxyl groups of glycerol and a second alcohol the head group esterified to the third hydroxyl of glycerol via a phosphodiester bond Other polar lipids are the sterols I Glycerophospholipids differ in the structure of their head group common glycerophospholipids are phosphatidylethanolamine and phosphatidylcholine The polar heads of the glycerophospholipids carry electric charges at pH near 7 I Chloroplast membranes are remarkably rich in galactolipids composed of a diacylglycerol with one or two linked galactose residues and sulfolipids diacylglycerols with a linked sulfonated sugar residue and thus a negatively charged head group Archaebacteria have unique membrane lipids with longchain alkyl groups etherlinked to glycerol at both ends and with sugar residues andor phosphate joined to the glycerol to provide a polar or charged head group These lipids are stable under the harsh conditions in which archaebacteria live The sphingolipids contain sphingosine a long chain aliphatic amino alcohol but no glycerol Sphingomyelin has in addition to phosphoric acid and choline two long hydrocarbon chains one contributed by a fatty acid and the other by sphingosine Three other classes of sphingolipids are cerebrosides globosides and gangliosides which contain sugar components Sterols have four fused rings and a hydroxyl group Cholesterol the major sterol in animals is both a structural component of membranes and precursor to a wide variety of steroids SUMMARY 103 Lipids as Signals Cofactors and Pigments I Some types of lipids although present in relatively small quantities play critical roles as cofactors or signals I Phosphatidylinositol bisphosphate is hydrolyzed to yield two intracellular messengers diacylglycerol and inositol 145 trisphosphate Phosphatidylinositol 345trisphosphate is a nucleation point for supramolecular protein complexes involved in biological signaling I Prostaglandins thromboxanes and leukotrienes the eicosanoids derived from arachidonate are extremely potent hormones I Steroid hormones derived from sterols serve as powerful biological signals such as the sex hormones I Vitamins D A E and K are fatsoluble compounds made up of isoprene units All play essential roles in the metabolism or physiology of animals Vitamin D is precursor to a hormone that regulates calcium metabolism Vitamin A furnishes the visual pigment of the vertebrate eye and is a regulator of gene expression during epithelial cell growth Vitamin E functions in the protection of membrane lipids from oxidative damage and vitamin K is essential in the bloodclotting process I Ubiquinones and plastoquinones also isoprenoid derivatives function as electron carriers in mitochondria and chloroplasts respectively I Dolichols activate and anchor sugars on cellular membranes for use in the synthesis of certain complex carbohydrates glycolipids and glycoproteins SUMMARY 104 Working with Lipids I In the determination of lipid composition the lipids are first extracted from tissues with organic solvents and separated by thinlayer gasliquid or highperformance liquid chromatography I Phospholipases specific for one of the bonds in a phospholipid can be used to generate simpler compounds for subsequent analysis I Individual lipids are identified by their chromatographic behavior their susceptibility to hydrolysis by specific enzymes or mass spectrometry SUMMARY 111 The Composition and Architecture of Membranes Biological membranes define cellular boundaries divide cells into discrete compartments organize complex reaction sequences and act in signal reception and energy transformations Membranes are composed of lipids and proteins in varying combinations particular to each species cell type and organelle The uid mosaic model describes features common to all biological membranes The lipid bilayer is the basic structural unit Fatty acyl chains of phospholipids and the steroid nucleus of sterols are oriented toward the interior of the bilayer their hydrophobic interactions stabilize the bilayer but give it exibility Peripheral proteins are loosely associated with the membrane through electrostatic interactions and hydrogen bonds or by covalently attached lipid anchors Integral proteins associate firmly with membranes by hydrophobic interactions between the lipid bilayer and their nonpolar amino acid side chains which are oriented toward the outside of the protein molecule Some membrane proteins span the lipid bilayer several times with hydrophobic sequences of about 20 amino acid residues forming transmembrane helices Detection of such hydrophobic sequences in proteins can be used to predict their secondary structure and transmembrane disposition Multistranded barrels are also common in integral membrane proteins Tyr and Trp residues of transmembrane proteins are commonly found at the lipidwater interface The lipids and proteins of membranes are inserted into the bilayer with specific sidedness thus membranes are structurally and function ally asymmetric Many membrane proteins contain covalently attached oligosaccharides Plasma membrane glycoproteins are always oriented with the carbohydratebearing domain on the extracellular surface SUMMARY 112 Membrane Dynamics Lipids in a biological membrane can exist in liquidordered or liquiddisordered states in the latter state thermal motion of acyl chains makes the interior of the bilayer uid Fluidity is affected by temperature fatty acid composition and sterol content Flip op diffusion of lipids between the inner and outer lea ets of a membrane is very slow except when specifically catalyzed by ippases Lipids and proteins can diffuse laterally within the plane of the membrane but this mobility is limited by interactions of membrane proteins with internal cytoskeletal structures and interactions of lipids with lipid rafts One class of lipid rafts consists of sphingolipids and cholesterol with a subset of membrane proteins that are GPIlinked or attached to several long chain fatty acyl moieties Caveolin is an integral membrane protein that associates with the inner lea et of the plasma membrane forcing it to curve inward to form caveolae probably involved in membrane transport and signaling Integrins are transmembrane proteins of the plasma membrane that act both to attach cells to each other and to carry messages between the extracellular matrix and the cytoplasm I Speci c proteins mediate the fusion of two membranes which accompanies processes such as viral invasion and endocytosis and exocytosis SUMMARY 113 Solute Transport across Membranes Movement of polar compounds and ions across biological membranes requires protein transporters Some transporters simply facilitate passive diffusion across the membrane from the side with higher concentration to the side with lower Others bring about active movement of solutes against an electrochemical gradient such transport must be coupled to a source of metabolic energy Carriers like enzymes show saturation and stereospecificity for their substrates Transport via these systems may be passive or active Primary active transport is driven by ATP or electrontransfer reactions secondary active transport by coupled ow of two solutes one of which often H or Na ows down its electrochemical gradient as the other is pulled up its gradient The GLUT transporters such as GLUTl of erythrocytes carry glucose into cells by facilitated diffusion These transporters are uniporters carrying only one substrate Symporters permit simultaneous passage of two substances in the same direction examples are the lactose transporter of E coli driven by the energy of a proton gradient lactoseH symport and the glucose transporter of intestinal epithelial cells driven by a Na gradient glucoseNa symport Antiporters mediate simultaneous passage of two substances in opposite directions examples are the chloride bicarbonate exchanger of erythrocytes and the ubiquitous Na K ATPase I In animal cells Na K ATPase maintains the differences in cytosolic and extracellular concentrations of Na and K and the resulting Na gradient is used as the energy source for a variety of secondary active transport processes I The Na K ATPase of the plasma membrane and the Ca2 transporters of the sarcoplasmic and endoplasmic reticulums the SERCA pumps are examples of Ptype ATPases they undergo reversible phosphorylation during their catalytic cycle and are inhibited by the phosphate analog vanadate Ftype ATPase proton pumps ATP synthases are central to energy conserving mechanisms in mitochondria and chloroplasts Vtype ATPases produce gradients of protons across some intracellular membranes including plant vacuolar membranes I ABC transporters carry a variety of substrates including many drugs out of cells using ATP as energy source I Ionophores are lipidsoluble molecules that bind speci c ions and carry them passively across membranes dissipating the energy of electrochemical ion gradients I Water moves across membranes through aquaporins I Ion channels provide hydrophilic pores through which select ions can diffuse moving down their electrical or chemical concentration gradients they are characteristically unsaturable and have very high ux rates Many ion channels are highly speci c for one ion and most are gated by either voltage or a ligand In bacterial K channels a selectivity filter provides ligands with the right geometry to replace the water of hydration of a K ion as it crosses the membrane Some K channels are voltage gated The acetylcholine receptorchannel is gated by acetylcholine which triggers subtle conformational changes that open and close the transmembrane path SUMMARY 121 Molecular Mechanisms of Signal Transduction All cells have speci c and highly sensitive signaltransducing mechanisms which have been conserved during evolution A wide variety of stimuli including hormones neurotransmitters and growth factors act through speci c protein receptors in the plasma membrane The receptors bind the signal molecule amplify the signal integrate it with input from other receptors and transmit it into the cell If the signal persists receptor desensitization reduces or ends the response Eukaryotic cells have six general types of signaling mechanisms gated ion channels receptor enzymes membrane proteins that act through G proteins nuclear proteins that bind steroids and act as transcription factors membrane proteins that attract and activate soluble protein kinases and adhesion receptors that carry information between the extracellular matrix and the cytoskeleton SUMMARY 122 Gated Ion Channels Ion channels gated by ligands or membrane potential are central to signaling in neurons and other cells The acetylcholine receptor of neurons and myocytes is a ligandgated ion channel The voltagegated Na and K channels of neuronal membranes carry the action potential along the axon as a wave of depolarization Na in ux followed by repolarization K ef ux The arrival of an action potential triggers neurotransmitter release from the presynaptic cell The neurotransmitter acetylcholine for example diffuses to the postsynaptic cell binds to specific receptors in the plasma membrane and triggers a change in Vm SUMMARY 123 Receptor Enzymes The insulin receptor is the prototype of receptor enzymes with Tyr kinase activity When insulin binds to its receptor each monomer of the receptor phosphorylates the chain of its partner activating the receptor s Tyr kinase activity The kinase catalyzes the phosphorylation of Tyr residues on other proteins such as IRSl P Tyr residues in IRSl serve as binding sites for proteins with SH2 domains Some of these proteins such as Grb2 have two or more proteinbinding domains and can serve as adaptors that bring two proteins into proximity Further proteinprotein interactions result in GTP binding to and activation of the Ras protein which in turn activates a protein kinase cascade that ends with the phosphorylation of target proteins in the cytosol and nucleus The result is specific metabolic changes and altered gene expression Several signals including atrial natriuretic factor and the intestinal peptide guanylin act through receptor enzymes with guanylyl cyclase activity The cGMP produced acts as a second messenger activating cGMPdependent protein kinase PKG This enzyme alters metabolism by phosphorylating specific enzyme targets Nitric oxide NO is a shortlived messenger that acts by stimulating a soluble guanylyl cyclase raising cGMP and stimulating PKG SUMMARY 124 G Protein Coupled Receptors and Second Messengers A large family of plasma membrane receptors with seven transmembrane segments act through heterotrimeric G proteins On ligand binding these receptors catalyze the exchange of GTP for GDP bound to an associated G protein forcing dissociation of the subunit of the G protein This subunit stimulates or inhibits the activity of a nearby membranebound enzyme changing the level of its second messenger product The adrenergic receptor binds epinephrine then through a stimulatory G protein GS activates adenylyl cyclase in the plasma membrane The cAMP produced by adenylyl cyclase is an intracellular second messenger that stimulates cAMP dependent protein kinase which mediates the effects of epinephrine by phosphorylating key proteins changing their enzymatic activities or structural features The cascade of events in which a single molecule of hormone activates a catalyst that in turn activates another catalyst and so on results in large signal amplification this is characteristic of most hormoneactivated systems Some receptors stimulate adenylyl cyclase through GS others inhibit it through Gi Thus cellular cAMP re ects the integrated input of two or more signals Cyclic AMP is eventually eliminated by cAMP phosphodiesterase and GS turns itself off by hydrolysis of its bound GTP to GDP When the epinephrine signal persists adrenergic receptor specific protein kinase and arrestin 2 temporarily desensitize the receptor and cause it to move into intracellular vesicles In some cases arrestin also acts as a scaffold protein bringing together protein components of a signaling pathway such as the MAPK cascade Some serpentine receptors are coupled to a plasma membrane phospholipase C that cleaves PIP2 to diacylglycerol and IP3 By opening Caz channels in the endoplasmic reticulum IP3 raises cytosolic Caz Diacylglycerol and Caz act together to activate protein kinase C which phosphorylates and changes the activity of specific cellular proteins Cellular Caz also regulates a number of other enzymes often through calmodulin SUMMARY 125 Multivalent Scaffold Proteins and Membrane Rafts I Many signaling proteins have domains that bind phosphorylated Tyr Ser or Thr residues in other proteins the binding specificity for each domain is determined by sequences that adjoin the phosphorylated residue I SH2 and PTB domains bind to proteins containing P Tyr residues other domains bind P Ser and P Thr residues in various contexts I Plextrin homology domains bind the membrane phospholipid PIP3 I Many signaling proteins are multivalent with several different binding modules By combining the substrate specificities of various protein kinases with the specificities of domains that bind phosphorylated Ser Thr or Tyr residues and with phosphatases that can rapidly inactivate a pathway cells create a large number of multiprotein signaling complexes I Membrane rafts and caveolae sequester groups of signaling proteins in small regions of the plasma membrane enhancing their interactions and making signaling more efficient SUMMARY 126 Signaling in Microorganisms and Plants Bacteria and unicellular eukaryotes have a variety of sensory systems that allow them to sample and respond to their environment In the twocomponent system a receptor His kinase senses the signal and autophosphory lates a His residue then phosphorylates the response regulator on an Asp residue Plants respond to many environmental stimuli and employ hormones and growth factors to coordinate the development and metabolic activities of their tissues Plant genomes encode hundreds of signaling proteins including some very similar to those used in signal transductions in mammalian cells Twocomponent signaling mechanisms common in bacteria have been acquired in altered forms by plants Cyanobacteria use typical twocomponent systems in the detection of chemical signals and light plants use related proteins which autophosphorylate on SerThr not His residues to detect ethylene Plant receptorlike kinases RLKs with an extracellular ligandbinding domain a single transmembrane segment and a cytosolic protein kinase domain participate in detecting a wide variety of stimuli including peptides that originate from pathogens brassinosteroid hormones selfincompatible pollen and developmental signals RLKs autophosphorylate SerThr residues then activate downstream proteins that in some cases are MAPK cascades The end result of many such signals is increased transcription of specific genes SUMMARY 12 7 Sensory Transduction in Vision Olfaction and Gustation I Vision olfaction and gustation in vertebrates employ serpentine receptors which act through heterotrimeric G proteins to change the Vm of the sensory neuron In rod and cone cells of the retina light activates rhodopsin which stimulates replacement of GDP by GTP on the G protein transducin The freed subunit of transducin activates cGMP phosphodiesterase which lowers cGMP and thus closes cGMPdependent ion channels in the outer segment of the neuron The resulting hyperpolarization of the rod or cone cell carries the signal to the next neuron in the pathway and eventually to the brain In olfactory neurons olfactory stimuli acting through serpentine receptors and G proteins trigger either an increase in cAMP by activating adenylyl cyclase or an increase in Caz by activating PLC These second messengers affect ion channels and thus the Vm Gustatory neurons have serpentine receptors that respond to tastants by altering cAMP which in turn changes Vm by gating ion channels There is a high degree of conservation of signaling proteins and transduction mechanisms across species SUMMARY 128 Regulation of Transcription by Steroid Hormones Steroid hormones enter cells and bind to specific receptor proteins The hormonereceptor complex binds specific regions of DNA the hormone response elements and regulates the expression of nearby genes by interacting with transcription factors Two other fasteracting mechanisms produce some of the effects of steroids Progesterone triggers a rapid drop in cAMP mediated by a plasma membrane receptor and binding of progesterone to the classic soluble steroid receptor activates a MAPK cascade SUMMARY 129 Regulation of the Cell Cycle by Protein Kinases I Progression through the cell cycle is regulated by the cyclindependent protein kinases CDKs which act at specific points in the cycle phosphorylating key proteins and modulating their activities The catalytic subunit of CDKs is inactive unless associated with the regulatory cyclin subunit I The activity of a cyclinCDK complex changes during the cell cycle through differential synthesis of CDKs specific degradation of cyclin phosphorylation and dephosphorylation of critical residues in CDKs and binding of inhibitory proteins to specific cyclinCDKs SUMMARY 1210 Oncogenes Tumor Suppressor Genes and Programmed Cell Death Oncogenes encode defective signaling proteins By continually giving the signal for cell division they lead to tumor formation Oncogenes are genetically dominant and may encode defective growth factors receptors G proteins protein kinases or nuclear regulators of transcription Tumor suppressor genes encode regulatory proteins that normally inhibit cell division mutations in these genes are genetically recessive but can lead to tumor formation Cancer is generally the result of an accumulation of mutations in oncogenes and tumor suppressor genes Apoptosis can be triggered by extracellular signals such as TNF through plasma membrane receptors SUMMARY 131 Bioenergetics and Thermodynamics I Living cells constantly perform work They require energy for maintaining their highly organized structures synthesizing cellular components generating electric currents and many other processes I Bioenergetics is the quantitative study of energy relationships and energy conversions in biological systems Biological energy transformations obey the laws of thermodynamics I All chemical reactions are in uenced by two forces the tendency to achieve the most stable bonding state for which enthalpy H is a useful expression and the tendency to achieve the highest degree of randomness expressed as entropy S The net driving force in a reaction is G the freeenergy change which represents the net effect of these two factors G H T S I The standard transformed freeenergy change G is a physical constant that is characteristic for a given reaction and can be calculated from the equilibrium constant for the reaction G RT ln K eq I The actual freeenergy change G is a variable that depends on G and on the concentrations of reactants and products G G RT ln productsreactants I When G is large and negative the reaction tends to go in the forward direction when G is large and positive the reaction tends to go in the reverse direction and when G O the system is at equilibrium I The freeenergy change for a reaction is independent of the pathway by which the reaction occurs Freeenergy changes are additive the net chemical reaction that results from successive reactions sharing a common intermediate has an overall free energy change that is the sum of the G values for the individual reactions SUMMARY 132 Phosphoryl Group Transfers and ATP ATP is the chemical link between catabolism and anabolism It is the energy currency of the living cell The exergonic conversion of ATP to ADP and Pi or to AMP and PPi is coupled to many endergonic reactions and processes Direct hydrolysis of ATP is the source of energy in the conformational changes that produce muscle contraction but in general it is not ATP hydrolysis but the transfer of a phosphoryl pyrophosphoryl or adenylyl group from ATP to a substrate or enzyme molecule that couples the energy of ATP breakdown to endergonic transformations of substrates Through these group transfer reactions ATP provides the energy for anabolic reactions including the synthesis of informational molecules and for the transport of molecules and ions across membranes against concentration gradients and electrical potential gradients Cells contain other metabolites with large negative free energies of hydrolysis including phosphoenolpyruvate 13 bisphosphoglycerate and phosphocreatine These highenergy compounds like ATP have a high phosphoryl group transfer potential they are good donors of the phosphoryl group Thioesters also have high free energies of hydrolysis Inorganic polyphosphate present in all cells may serve as a reservoir of phosphoryl groups with high group transfer potential SUMMARY 133 Biological OxidationReduction Reactions I In many organisms a central energyconserving process is the stepwise oxidation of glucose to CO2 in which some of the energy of oxidation is conserved in ATP as electrons are passed to O2 Biological oxidationreduction reactions can be described in terms of two halfreactions each with a characteristic standard reduction potential E When two electrochemical halfcells each containing the components of a halfreaction are connected electrons tend to ow to the halfcell with the higher reduction potential The strength of this tendency is proportional to the difference between the two reduction potentials E and is a function of the concentrations of oxidized and reduced species The standard freeenergy change for an oxidationreduction reaction is directly proportional to the difference in standard reduction potentials of the two halfcells G n E Many biological oxidation reactions are dehydrogenations in which one or two hydrogen atoms H e are transferred from a substrate to a hydrogen acceptor Oxidationreduction reactions in living cells involve specialized electron carriers NAD and NADP are the freely diffusible coenzymes of many dehydrogenases Both NAD and NADP accept two electrons and one proton NAD and NADP are bound to dehydrogenases in a widely conserved structural motif called the Rossmann fold FAD and FMN the avin nucleotides serve as tightly bound prosthetic groups of avoproteins They can accept either one or two electrons Flavoproteins also serve as light receptors in cryptochromes and photolyases SUMMARY 141 Glycolysis Glycolysis is a nearuniversal pathway by which a glucose molecule is oxidized to two molecules of pyruvate with energy conserved as ATP and NADH All ten glycolytic enzymes are in the cytosol and all ten intermediates are phosphorylated compounds of three or six carbons In the preparatory phase of glycolysis ATP is invested to convert glucose to fructose 16bisphosphate The bond between C 3 and C4 is then broken to yield two molecules of triose phosphate In the payoff phase each of the two molecules of glyceraldehyde 3phosphate derived from glucose undergoes oxidation at C l the energy of this oxidation reaction is conserved in the formation of one NADH and two ATP per triose phosphate oxidized The net equation for the overall process is Glucose 2NAD 2ADP 2Pi 88n2pyruvate 2NADH 2H 2ATP 2H2O Glycolysis is tightly regulated in coordination with other energyyielding pathways to assure a steady supply of ATP Hexokinase PFKl and pyruvate kinase are all subject to allosteric regulation that controls the ow of carbon through the pathway and maintains constant levels of metabolic intermediates SUMMARY 142 Feeder Pathways for Glycolysis I Glycogen and starch polymeric storage forms of glucose enter glycolysis in a twostep process Phosphorolytic cleavage of a glucose residue from an end of the polymer forming glucose lphosphate is catalyzed by glycogen phosphorylase or starch phosphorylase Phosphoglucomutase then converts the glucose lphosphate to glucose 6phosphate which can enter glycolysis I Ingested polysaccharides and disaccharides are converted to monosaccharides by intestinal hydrolytic enzymes and the monosaccharides then enter intestinal cells and are transported to the liver or other tissues I A variety of Dhexoses including fructose galactose and mannose can be funneled into glycolysis Each is phosphorylated and converted to either glucose 6phosphate or fructose 6phosphate I Conversion of galactose lphosphate to glucose lphosphate involves two nucleotide derivatives UDPgalactose and UDP glucose Genetic de fects in any of the three enzymes that catalyze conversion of galactose to glucose lphosphate result in galactosemias of varying severity SUMMARY 143 Fates of Pyruvate under Anaerobic Conditions Fermentation The NADH formed in glycolysis must be recycled to regenerate NAD which is required as an electron acceptor in the first step of the payoff phase Under aerobic conditions electrons pass from NADH to O2 in mitochondrial respiration Under anaerobic or hypoxic conditions many organisms regenerate NAD by transferring electrons from NADH to pyruvate forming lactate Other organisms such as yeast regenerate NAD by reducing pyruvate to ethanol and CO In these anaerobic processes fermentations there is no net oxidation or reduction of the carbons of glucose I A variety of microorganisms can ferment sugar in fresh foods resulting in changes in pH taste and texture and preserving food from spoilage Fermentations are used in industry to produce a wide variety of commercially valuable organic compounds from inexpensive starting materials SUMMARY 144 Gluconeogenesis Gluconeogenesis is a ubiquitous multistep process in which pyruvate or a related threecarbon compound lactate alanine is converted to glucose Seven of the steps in gluconeogenesis are catalyzed by the same enzymes used in glycolysis these are the reversible reactions Three irreversible steps in the glycolytic pathway are bypassed by reactions catalyzed by gluconeogenic enzymes 1 conversion of pyruvate to PEP via oxaloacetate catalyzed by pyruvate carboxylase and PEP carboxykinase 2 dephosphorylation of fructose 16bisphosphate by FBPasel and 3 dephosphorylation of glucose 6phosphate by glucose 6phosphatase Formation of one molecule of glucose from pyruvate requires 4 ATP 2 GTP and 2 NADH it is expensive In mammals gluconeogenesis in the liver and kidney provides glucose for use by the brain muscles and erythrocytes Pyruvate carboxylase is stimulated by acetylCoA increasing the rate of gluconeogenesis when the cell already has adequate supplies of other substrates fatty acids for energy production Animals cannot convert acetylCoA derived from fatty acids into glucose plants and microorganisms can Glycolysis and gluconeogenesis are reciprocally regulated to prevent wasteful operation of both pathways at the same time SUMMARY 145 Pentose Phosphate Pathway of Glucose Oxidation The oxidative pentose phosphate pathway phosphogluconate pathway or hexose monophosphate pathway brings about oxidation and decarboxylation at C1 of glucose 6phosphate reducing NADP to NADPH and producing pentose phosphates NADPH provides reducing power for biosynthetic reactions and ribose 5phosphate is a precursor for nucleotide and nucleic acid synthesis Rapidly growing tissues and tissues carrying out active biosynthesis of fatty acids cholesterol or steroid hormones send more glucose 6phosphate through the pentose phosphate pathway than do tissues with less demand for pentose phosphates and reducing power The first phase of the pentose phosphate pathway consists of two oxidations that convert glucose 6phosphate to ribulose 5 phosphate and reduce NADP to NADPH The second phase comprises nonoxidative steps that convert pentose phosphates to glucose 6phosphate which begins the cycle again I In the second phase transaldolase with TPP as cofactor and transketolase catalyze the interconversion of three four five six and sevencarbon sugars with the reversible conversion of six pentose phosphates to five hexose phosphates In the carbonassimilating reactions of photosynthesis the same enzymes catalyze the reverse process called the reductive pentose phosphate pathway conversion of five hexose phosphates to six pentose phosphates I A genetic defect in transketolase that lowers its affinity for TPP exacerbates the Wernicke Korsakoff syndrome I Entry of glucose 6phosphate either into glycolysis or into the pentose phosphate pathway is largely determined by the relative concentrations of NADP and NADPH SUMMARY 151 The Metabolism of Glycogen in Animals Glycogen is stored in muscle and liver as large particles Contained within the particles are the enzymes that metabolize glycogen as well as regulatory enzymes Glycogen phosphorylase catalyzes phosphorolytic cleavage at the nonreducing ends of glycogen chains producing glucose 1 phosphate The debranching enzyme transfers branches onto main chains and releases the residue at the ln6 branch as free glucose Phosphoglucomutase interconverts glucose lphosphate and glucose 6phosphate Glucose 6phosphate can enter glycolysis or in liver can be converted to free glucose by glucose 6phosphatase in the endoplasmic reticulum then released to replenish blood glucose The sugar nucleotide UDPglucose donates glucose residues to the nonreducing end of glycogen in the reaction catalyzed by glycogen synthase A separate branching enzyme produces the ln6 linkages at branch points New glycogen particles begin with the auto catalytic formation of a glycosidic bond between the glucose of UDPglucose and a Tyr residue in the protein glycogenin followed by addition of several glucose residues to form a primer that can be acted upon by glycogen synthase SUMMARY 152 Regulation of Metabolic Pathways In a metabolically active cell in a steady state intermediates are formed and consumed at equal rates When a perturbation alters the rate of formation or consumption of a metabolite compensating changes in enzyme activities return the system to the steady state Regulatory mechanisms maintain nearly constant levels of key metabolites such as ATP and NADH in cells and glucose in the blood while matching the use or storage of glycogen to the organism s changing needs In multistep processes such as glycolysis certain reactions are essentially at equilibrium in the steady state the rates of these substratelimited reactions rise and fall with substrate concentration Other reactions are far from equilibrium their rates are too slow to produce instant equilibration of substrate and product These enzymelimited reactions are often highly exergonic and therefore metabolically irreversible and the enzymes that catalyze them are commonly the points at which ux through the pathway is regulated The activity of an enzyme can be regulated by changing the rate of its synthesis or degradation by allosteric or covalent alteration of existing enzyme molecules or by separating the enzyme from its substrate in subcellular compartments Fast metabolic adjustments on the time scale of seconds or less at the intracellular level are generally allosteric The effects of hormones and growth factors are generally slower seconds to hours and are commonly achieved by covalent modification or changes in enzyme synthesis SUMMARY 153 Coordinated Regulation of Glycolysis and Gluconeogenesis I Three glycolytic enzymes are subject to allosteric regulation hexokinase IV phosphofructokinasel PFKl and pyruvate kinase I Hexokinase IV glucokinase is sequestered in the nucleus of the hepatocyte but is released when the cytosolic glucose concentration rises I PFKl is allosterically inhibited by ATP and citrate In most mammalian tissues including liver PFKl is allosterically activated by fructose 26bisphosphate I Pyruvate kinase is allosterically inhibited by ATP and the liver isozyme is inhibited by cAMPdependent phosphorylation I Gluconeogenesis is regulated at the level of pyruvate carboxylase which is activated by acetylCoA and FBPasel which is inhibited by fructose 26bisphosphate and AMP I To limit futile cycling between glycolysis and gluconeogenesis the two pathways are under reciprocal allosteric control mainly achieved by the opposite effects of fructose 26 bisphosphate on PFKl and FBPasel I Glucagon or epinephrine decreases fructose 26bisphosphate The hormones do this by raising cAMP and bringing about phosphorylation of the bifunctional enzyme that makes and breaks down fructose 26 bisphosphate Phosphorylation inactivates PFK2 and activates FBPase2 leading to breakdown of fructose 26bisphosphate Insulin increases fructose 26 bisphosphate by activating a phosphoprotein phosphatase that dephosphorylates activates PFK2 SUMMARY 154 Coordinated Regulation of Glycogen Synthesis and Breakdown Glycogen phosphorylase is activated in response to glucagon or epinephrine which raise cAMP and activate PKA PKA phosphorylates and activates phosphorylase kinase which converts glycogen phosphorylase b to its active a form Phosphoprotein phosphatase 1 PPl reverses the phosphorylation of glycogen phosphorylase a inactivating it Glucose binds to the liver isozyme of glycogen phosphorylase a favoring its dephosphorylation and inactivation Glycogen synthase a is inactivated by phosphorylation catalyzed by GSK3 Insulin blocks GSK3 PPl which is activated by insulin reverses the inhibition by dephosphorylating glycogen synthase b Insulin increases glucose uptake into myocytes and adipocytes by triggering movement of the glucose transporter GLUT4 to the plasma membrane Insulin stimulates the synthesis of hexokinases II and IV PFKl pyruvate kinase and several enzymes involved in lipid synthesis Insulin stimulates glycogen synthesis in muscle and liver IIn liver glucagon stimulates glycogen breakdown and gluconeogenesis while blocking glycolysis thereby sparing glucose for export to the brain and other tissues In muscle epinephrine stimulates glycogen breakdown and glycolysis providing ATP to support contraction SUMMARY 155 Analysis of Metabolic Control I Metabolic control analysis shows that control of the rate of metabolite ux through a pathway is distributed among several of the enzymes in that path I The ux control coefficient C is an experimentally determined measure of the effect of an enzyme s concentration on ux through a multienzyme pathway It is characteristic of the whole system not intrinsic to the enzyme I The elasticity coefficient of an enzyme is an experimentally determined measure of how responsive the enzyme is to changes in the concentration of a metabolite or regulator molecule I The response coefficient R is the expression for the experimentally determined change in ux through a pathway in response to a regulatory hormone or second messenger It is afunctionofCand R C I Some regulated enzymes control the ux through a pathway while others rebalance the level of metabolites in response to the change in ux This latter rebalancing activity is regulation the former activity is control I Metabolic control analysis predicts that ux toward a desired product is most effectively increased by raising the concentration of all enzymes in the pathway SUMMARY 161 Production of AcetylCoA Activated Acetate Pyruvate the product of glycolysis is converted to acetylCoA the starting material for the citric acid cycle by the pyruvate dehydrogenase complex The PDH complex is composed of multiple copies of three enzymes pyruvate dehydrogenase E1 with its bound cofactor TPP dihydrolipoyl transacetylase E2 with its covalently bound lipoyl group and dihydrolipoyl dehydrogenase E3 with its cofactors FAD and NAD E1 catalyzes first the decarboxylation of pyruvate producing hydroxyethylTPP and then the oxidation of the hydroxyethyl group to an acetyl group The electrons from this oxidation reduce the disulfide of lipoate bound to E2 and the acetyl group is transferred into thioester linkage with one OSH group of reduced lipoate E2 catalyzes the transfer of the acetyl group to coenzyme A forming acetylCoA E3 catalyzes the regeneration of the disulfide oxidized form of lipoate electrons pass first to FAD then to NAD The long lipoyllysine arm swings from the active site of E1 to E2 to E3 tethering the intermediates to the enzyme complex to allow substrate channeling I The organization of the PDH complex is very similar to that of the enzyme complexes that catalyze the oxidation of ketoglutarate and the branchedchain keto acids SUMMARY 162 Reactions of the Citric Acid Cycle I The citric acid cycle Krebs cycle TCA cycle is a nearly universal central catabolic pathway in which compounds derived from the break down of carbohydrates fats and proteins are oxidized to CO2 with most of the energy of oxidation temporarily held in the electron carriers FADH2 and NADH During aerobic metabolism these electrons are transferred to O2 and the energy of electron ow is trapped as ATP I AcetylCoA enters the citric acid cycle in the mitochondria of eukaryotes the cytosol of prokaryotes as citrate synthase catalyzes its condensation with oxaloacetate to form citrate I In seven sequential reactions including two decarboxylations the citric acid cycle converts citrate to oxaloacetate and releases two C02 The pathway is cyclic in that the intermediates of the cycle are not used up for each oxalo acetate consumed in the path one is produced I For each acetylCoA oxidized by the citric acid cycle the energy gain consists of three molecules of NADH one FADH2 and one nucleoside triphosphate either ATP or GTP I Besides acetylCoA any compound that gives rise to a four or fivecarbon intermediate of the citric acid cycle for example the break down products of many amino acids can be oxidized by the cycle I The citric acid cycle is amphibolic serving in both catabolism and anabolism cycle inter mediates can be drawn off and used as the starting material for a variety of biosynthetic products I When intermediates are shunted from the citric acid cycle to other pathways they are replenished by several anaplerotic reactions which produce fourcarbon intermediates by carboxylation of threecarbon compounds these reactions are catalyzed by pyruvate carboxylase PEP carboxykinase PEP carboxylase and malic enzyme Enzymes that catalyze carboxylations commonly employ biotin to activate CO2 and to carry it to acceptors such as pyruvate or phosphoenolpyruvate SUMMARY 163 Regulation of the Citric Acid Cycle The overall rate of the citric acid cycle is controlled by the rate of conversion of pyruvate to acetylCoA and by the ux through citrate synthase isocitrate dehydrogenase and ketoglutarate dehydrogenase These uxes are largely determined by the concentrations of substrates and products the end products ATP and NADH are inhibitory and the substrates NAD and ADP are stimulatory The production of acetylCoA for the citric acid cycle by the PDH complex is inhibited allosterically by metabolites that signal a sufficiency of metabolic energy ATP acetyl CoA NADH and fatty acids and stimulated by metabolites that indicate a reduced energy supply AMP NAD CoA SUMMARY 164 The Glyoxylate Cycle The glyoxylate cycle is active in the germinating seeds of some plants and in certain microorganisms that can live on acetate as the sole carbon source In plants the pathway takes place in glyoxysomes in seedlings It involves several citric acid cycle enzymes and two additional enzymes isocitrate lyase and malate synthase In the glyoxylate cycle the bypassing of the two decarboxylation steps of the citric acid cycle makes possible the net formation of succinate oxaloacetate and other cycle intermediates from acetylCoA Oxaloacetate thus formed can be used to synthesize glucose via gluconeogenesis The partitioning of isocitrate between the citric acid cycle and the glyoxylate cycle is controlled at the level of isocitrate dehydrogenase which is regulated by reversible phosphorylation Vertebrates lack the glyoxylate cycle and cannot synthesize glucose from acetate or the fatty acids that give rise to acetyl CoA SUMMARY l7l Digestion Mobilization and Transport of Fats I The fatty acids of triacylglycerols furnish a large fraction of the oxidative energy in animals Dietary triacylglycerols are emulsified in the small intestine by bile salts hydrolyzed by intestinal lipases absorbed by intestinal epithelial cells reconverted into triacylglycerols then formed into chylomicrons by combination with specific apolipoproteins Chylomicrons deliver triacylglycerols to tissues where lipoprotein lipase releases free fatty acids for entry into cells Triacylglycerols stored in adipose tissue are mobilized by a hormonesensitive triacylglycerol lipase The released fatty acids bind to serum albumin and are carried in the blood to the heart skeletal muscle and other tissues that use fatty acids for fuel Once inside cells fatty acids are activated at the outer mitochondrial membrane by conversion to fatty acyl CoA thioesters Fatty acyl CoA to be oxidized enters mitochondria in three steps via the carnitine shuttle SUMMARY 172 Oxidation of Fatty Acids I In the first stage of oxidation four reactions remove each acetylCoA unit from the carboxyl end of a saturated fatty acyl CoA l dehydrogenation of the and carbons C2 and C3 by FADlinked acyl CoA dehydrogenases 2 hydration of the resulting trans 2 double bond by enoylCoA hydratase 3 dehydrogenation of the resulting L hydroxyacylCoA by NADlinked hydroxyacylCoA dehydrogenase and4 CoArequiring cleavage of the resulting ketoacylCoA by thiolase to form acetylCoA and a fatty acyl CoA shortened by two carbons The shortened fatty acyl CoA then reenters the sequence In the second stage of fatty acid oxidation the acetylCoA is oxidized to CO in the citric acid 2 cycle A large fraction of the theoretical yieldof free energy from fatty acid oxidation is recovered as ATP by oxidative phosphorylation the final stage of the oxidative pathway MalonylCoA an early intermediate of fatty acid synthesis inhibits carnitine acyltransferase I preventing fatty acid entry into mitochondria This blocks fatty acid breakdown while synthesis is occurring Genetic defects in the mediumchain acyl CoA dehydrogenase result in serious human disease as do mutations in other components of the oxidation system Oxidation of unsaturated fatty acids requires two additional enzymes enoylCoA isomerase and 24dienoylCoA reductase Oddnumber fatty acids are oxidized by the oxidation pathway to yield acetylCoA and a molecule of propionylCoA This is carboxylated to methylmalonylCoA which is isomerized to succinylCoA in a reaction catalyzed by methylmalonylCoA mutase an enzyme requiring coenzyme B12 Peroxisomes of plants and animals and glyoxysomes of plants carry out oxidation in four steps similar to those of the mitochondrial pathway in animals The first oxidation step however transfers electrons directly to O2 generating H2O2 Peroxisomes of animal tissues specialize in the oxidation of verylongchain fatty acids and branched fatty acids In glyoxysomes in germinating seeds oxidation is one step in the conversion of stored lipids into a variety of intermediates and products I The reactions of oxidation occurring in the endoplasmic reticulum produce dicarboxylic fatty acyl intermediates which can undergo oxidation at either end to yield short dicarboxylic acids such as succinate SUMMARY 173 Ketone Bodies I The ketone bodies acetone acetoacetate and D hydroxybutyrate are formed in the liver The latter two compounds serve as fuel molecules in extrahepatic tissues through oxidation to acetylCoA and entry into the citric acid cycle I Overproduction of ketone bodies in uncontrolled diabetes or severely reduced calorie intake can lead to acidosis or ketosis SUMMARY 181 Metabolic Fates of Amino Groups I Humans derive a small fraction of their oxidative energy from the catabolism of amino acids Amino acids are derived from the normal breakdown recycling of cellular proteins degradation of ingested proteins and breakdown of body proteins in lieu of other fuel sources during starvation or in uncontrolled diabetes mellitus Proteases degrade ingested proteins in the stomach and small intestine Most proteases are initially synthesized as inactive zymogens An early step in the catabolism of amino acids is the separation of the amino group from the carbon skeleton In most cases the amino group is transferred to ketoglutarate to form glutamate This transamination reaction requires the coenzyme pyridoxal phosphate Glutamate is transported to liver mitochondria where glutamate dehydrogenase liberates the amino group as ammonium ion NH 4 Ammonia formed in other tissues is transported to the liver as the amide nitrogen of glutamine or in transport from skeletal muscle as the amino group of alanine The pyruvate produced by deamination of alanine in the liver is converted to glucose which is transported back to muscle as part of the glucosealanine cycle SUMMARY 182 Nitrogen Excretion and the Urea Cycle Ammonia is highly toxic to animal tissues In the urea cycle ornithine combines with ammonia in the form of carbamoyl phosphate to form citrulline A second amino group is transferred to citrulline from aspartate to form arginine the immediate precursor of urea Arginase catalyzes hydrolysis of arginine to urea and ornithine thus ornithine is regenerated in each turn of the cycle The urea cycle results in a net conversion of oxaloacetate to fumarate both of which are intermediates in the citric acid cycle The two cycles are thus interconnected The activity of the urea cycle is regulated at the level of enzyme synthesis and by allosteric regulation of the enzyme that catalyzes the formation of carbamoyl phosphate SUMMARY 183 Pathways of Amino Acid Degradation I After removal of their amino groups the carbon skeletons of amino acids undergo oxidation to compounds that can enter the citric acid cycle for oxidation to CO2 and H20 The reactions of these pathways require a number of cofactors including tetrahydrofolate and Sadenosylmethionine in onecarbon transfer reactions and tetrahydrobiopterin in the oxidation of phenylalanine by phenylalanine hydroxylase I Depending on their degradative end product some amino acids can be converted to ketone bodies some to glucose and some to both Thus amino acid degradation is integrated into intermediary metabolism and can be critical to survival under conditions in which amino acids are a significant source of metabolic energy I The carbon skeletons of amino acids enter the citric acid cycle through five intermediates acetylCoA ketoglutarate succinylCoA fumarate and oxaloacetate Some are also degraded to pyruvate which can be converted to either acetylCoA or oxaloacetate The amino acids producing pyruvate are alanine cysteine glycine serine threonine and tryptophan Leucine lysine phenylalanine and tryptophan yield acetylCoA via acetoacetylCoA Isoleucine leucine threonine and tryptophan also form acetylCoA directly Arginine glutamate glutamine histidine and proline produce ketoglutarate isoleucine methionine threonine and valine produce succinylCoA four carbon atoms of phenylalanine and tyrosine give rise to fumarate and asparagine and aspartate produce oxaloacetate I The branchedchain amino acids isoleucine leucine and valine unlike the other amino acids are degraded only in extrahepatic tissues A number of serious human diseases can be traced to genetic defects in the enzymes of amino acid catabolism SUMMARY 191 ElectronTransfer Reactions in Mitochondria Chemiosmotic theory provides the intellectual framework for understanding many biological energy transductions including oxidative phosphorylation and photophosphorylation The mechanism of energy coupling is similar in both cases the energy of electron ow is conserved by the concomitant pumping of protons across the membrane producing an electrochemical gradient the protonmotive force In mitochondria hydride ions removed from substrates by NADlinked dehydrogenases donate electrons to the respiratory electrontransfer chain which transfers the electrons to molecular 02 reducing it to H20 Shuttle systems convey reducing equivalents from cytosolic NADH to mitochondrial NADH Reducing equivalents from all NADlinked dehydrogenations are transferred to mito chondrial NADH dehydrogenase Complex I Reducing equivalents are then passed through a series of FeS centers to ubiquinone which transfers the electrons to cytochrome b the first carrier in Complex III In this complex electrons take two separate paths through two btype cytochromes and cytochrome cl to an FeS center The FeS center passes electrons one at a time through cytochrome c and into Complex IV cytochrome oxidase This coppercontaining enzyme which also contains cytochromes a and a3 accumulates electrons then passes them to 02 reducing it to H20 I Some electrons enter this chain of carriers through alternative paths Succinate is oxidized by succinate dehydrogenase Complex II which contains a avoprotein that passes electrons through several FeS centers to ubiquinone Electrons derived from the oxidation of fatty acids pass to ubiquinone via the electrontransferring avoprotein I Plants also have an alternative cyanideresistant NADH oxidation pathway SUMMARY 192 ATP Synthesis I The ow of electrons through Complexes I III and IV results in pumping of protons across the inner mitochondrial membrane making the matrix alkaline relative to the intermembrane space This proton gradient provides the energy in the form of the protonmotive force for ATP synthesis from ADP and Pi by ATP synthase F0 F1 complex in the inner membrane ATP synthase carries out rotational catalysis in which the ow of protons through F0 causes each of three nucleotide binding sites in F1 to cycle from ADP Pi bound to ATPbound to empty conformations ATP formation on the enzyme requires little energy the role of the protonmotive force is to push ATP from its binding site on the synthase The ratio of ATP synthesized per 12 02 reduced to H20 the P0 ratio is about 25 when elec trons enter the respiratory chain at Complex I and 15 when electrons enter at CoQ Energy conserved in a proton gradient can drive solute transport uphill across a membrane The inner mitochondrial membrane is impermeable to NADH and NAD but NADH equivalents are moved from the cytosol to the matrix by either of two shuttles NADH equivalents moved in by the malateaspartate shuttle enter the respiratory chain at Complex I and yield a P0 ratio of 25 those moved in by the glycerol 3phosphate shuttle enter at CoQ and give a P0 ratio of 15 SUMMARY 193 Regulation of Oxidative Phosphorylation I 0xidative phosphorylation is regulated by cellular energy demands The intracellular ADP and the massaction ratio ATPADPPi are measures of a cell s energy status In ischemic oxygendeprived cells a protein inhibitor blocks ATP hydrolysis by the ATP synthase operating in reverse preventing a drastic drop in ATP In brown fat which is specialized for the production of metabolic heat electron transfer is uncoupled from ATP synthesis and the energy of fatty acid oxidation is dissipated as heat ATP and ADP concentrations set the rate of electron transfer through the respiratory chain via a series of interlocking controls on respiration glycolysis and the citric acid cycle SUMMARY 194 Mitochondrial Genes Their Origin and the Effects of Mutations I A small proportion of human mitochondrial proteins 13 proteins are encoded in the mitochondrial genome and synthesized within mitochondria About 900 mitochondrial proteins are encoded in nuclear genes and imported into mitochondria after their synthesis I Mutations in the genes that encode components of the respiratory chain whether in the mitochondrial genes or in the nuclear genes that encode mitochondrial proteins cause a variety of human diseases which often affect muscle and brain most severely I Mitochondria most likely arose from aerobic prokaryotes that entered into an endosymbiotic relationship with ancestral eukaryotes SUMMARY 195 The Role of Mitochondria in Apoptosis and Oxidative Stress I Mitochondrial cytochrome c released into the cytosol participates in activation of one of the proteases caspase 9 involved in apoptosis I Reactive oxygen species produced in mitochondria are inactivated by a set of protective enzymes including superoxide dismutase and glutathione peroxidase SUMMARY 196 General Features of Photophosphorylation I The light reactions of photosynthesis are those directly dependent on the absorption of light the resulting photochemistry takes electrons from H20 and drives them through a series of membranebound carriers producing NADPH and ATP I The carbonassimilation reactions of photosynthesis reduce C02 with electrons from NADPH and energy from ATP SUMMARY 19 7 Light Absorption Photophosphorylation in the chloroplasts of green plants and in cyanobacteria involves electron ow through a series of membranebound carriers In the light reactions of plants absorption of a photon excites chlorophyll molecules and other accessory pigments which funnel the energy into reaction centers in the thylakoid membranes In the reaction centers photo excitation results in a charge separation that produces a strong electron donor reducing agent and a strong electron acceptor SUMMARY 198 The Central Photochemical Event LightDriven Electron Flow I Bacteria have a single reaction center in purple bacteria it is of the pheophytinquinone type and in green sulfur bacteria the FeS type I Structural studies of the reaction center of a purple bacterium have provided information about lightdriven electron ow from an excited special pair of chlorophyll molecules through pheophytin to quinones Electrons then pass from quinones through the cytochrome bcl complex and back to the photoreaction center I An alternative path in green sulfur bacteria sends electrons from reduced quinones to N AD Cyanobacteria and plants have two different photoreaction centers arranged in tandem Plant photosystem I passes electrons from its excited reaction center P700 through a series of carriers to ferredoxin which then reduces NADP to NADPH The reaction center of plant photosystem II P680 passes electrons to plastoquinone and the electrons lost from P680 are replaced by electrons from H20 electron donors other than H20 are used in other organisms The lightdriven splitting of H20 is catalyzed by a Mncontaining protein complex 02 is produced The reduced plastoquinone carries electrons to the cytochrome b6 f complex from here they pass to plastocyanin and then to P700 to replace those lost during its photoexcitation Electron ow through the cytochrome b6 f complex drives protons across the plasma membrane creating a protonmotive force that provides the energy for ATP synthesis by an ATP synthase SUMMARY 199 ATP Synthesis by Photophosphorylation In plants both the watersplitting reaction and electron ow through the cytochrome b6 f complex are accompanied by proton pumping across the thylakoid membrane The protonmotive force thus created drives ATP synthesis by a CFOCF1 complex similar to the mitochondrial F0 F1 complex Flow of electrons through the photosystems produces NADPH and ATP in the ratio of about 23 A second type of electron ow cyclic ow produces ATP only and allows variability in the proportions of NADPH and ATP formed The localization of PS1 and PSII between the granal and stromal lamellae can change and is indirectly controlled by light intensity optimizing the distribution of excitons between PSI and PSII for efficient energy capture Chloroplasts like mitochondria evolved from bacteria living endosymbiotically within early eukaryotic cells The ATP synthases of eubacteria cyanobacteria mitochondria and chloroplasts share a common evolutionary precursor and a common enzymatic mechanism Many photosynthetic microorganisms obtain electrons for photosynthesis not from water but from donors such as H2S SUMMARY 201 Photosynthetic Carbohydrate Synthesis Photosynthesis in vascular plants takes place in chloroplasts In the C02assimilating reactions the Calvin cycle ATP and NADPH are used to reduce C02 to triose phosphates These reactions occur in three stages the fixation reaction itself catalyzed by rubisco reduction of the resulting 3phosphoglycerate to glyceraldehyde 3phosphate and regeneration of ribulose 15bisphosphate from triose phosphates Rubisco condenses C02 with ribulose 15bisphosphate forming an unstable hexose bisphosphate that splits into two molecules of 3phosphoglycerate Rubisco is activated by covalent modification carbamoylation of Lyszol catalyzed by rubisco activase and is inhibited by a natural transitionstate analog whose concentration rises in the dark and falls during daylight Stromal isozymes of the glycolytic enzymes catalyze reduction of 3phosphoglycerate to glyceraldehyde 3phosphate each molecule reduced requires one ATP and one NADPH Stromal enzymes including transketolase and transaldolase rearrange the carbon skeletons of triose phosphates generating intermediates of three four five six and seven carbons and eventually yielding pentose phosphates The pentose phosphates are converted to ribulose 5phosphate then phosphorylated to ribulose 15bisphosphate to complete the Calvin cycle The cost of fixing three CO2 into one triose phosphate is nine ATP and six NADPH which are provided by the light dependent reactions of photosynthesis An antiporter in the inner chloroplast membrane exchanges Pi in the cytosol for 3phosphoglycerate or dihydroxyacetone phosphate produced by C02 assimilation in the stroma Oxidation of dihydroxyacetone phos phate in the cytosol generates ATP and NADH thus moving ATP and reducing equivalents from the chloroplast to the cytosol Four enzymes of the Calvin cycle are activated indirectly by light and are inactive in the dark so that hexose synthesis does not compete with glycolysis which is required to provide energy in the dark SUMMARY 202 Photorespiration and the C4 and CAM Pathways When rubisco uses 02 rather than CO2 as substrate the 2phosphoglycolate so formed is disposed of in an oxygendependent pathway The result is increased consumption of O2 photorespiration or more accurately the oxidative photosynthetic carbon cycle or C2 cycle The 2phosphoglycolate is converted to glyoxylate to glycine and then to serine in a pathway that involves enzymes in the chloroplast stroma the peroxisome and the mitochondrion In C4 plants the carbonassimilation pathway minimizes photorespiration CO2 is first fixed in mesophyll cells into a four carbon compound which passes into bundlesheath cells and releases CO2 in high concentrations The released CO2 is fixed by rubisco and the remaining reactions of the Calvin cycle occur as in C3 plants In CAM plants CO2 is fixed into malate in the dark and stored in vacuoles until daylight when the stomata are closed minimizing water loss and malate serves as a source of CO2 for rubisco SUMMARY 203 Biosynthesis of Starch and Sucrose Starch synthase in chloroplasts and amyloplasts catalyzes the addition of single glucose residues donated by ADPglucose to the reducing end of a starch molecule by a twostep insertion mechanism Branches in amylopectin are introduced by a second enzyme Sucrose is synthesized in the cytosol in two steps from UDPglucose and fructose lphosphate The partitioning of triose phosphates between sucrose synthesis and starch synthesis is regulated by fructose 26bisphosphate F26BP an allosteric effector of the enzymes that determine the level of fructose 6phosphate F26BP concentration varies inversely with the rate of photosynthesis and F26BP inhibits the synthesis of fructose 6phosphate the precursor to sucrose SUMMARY 204 Synthesis of Cell Wall Polysaccharides Plant Cellulose and Bacterial Peptidoglycan Cellulose synthesis takes place in terminal complexes rosettes in the plasma membrane Each cellulose chain begins as a sitosterol dextrin formed inside the cell It then ips to the outside where the oligosaccharide portion is transferred to cellulose synthase in the rosette and is then extended Each rosette produces 36 separate cellulose chains simultaneously and in parallel The chains crystallize into one of the microfibrils that form the cell wall Synthesis of the bacterial cell wall peptidoglycan also involves lipidlinked oligosaccharides formed inside the cell and ipped to the outside for assembly SUMMARY 205 Integration of Carbohydrate Metabolism in the Plant Cell Plants can synthesize sugars from acetylCoA the product of fatty acid breakdown by the combined actions of the glyoxylate cycle and gluconeogenesis The individual pathways of carbohydrate metabolism in plants overlap extensively they share pools of common intermediates including hexose phosphates pentose phosphates and triose phosphates Transporters in the membranes of chloroplasts mitochondria amyloplasts and peroxisomes mediate the movement of sugar phosphates between organelles The direction of metabolite ow through the pools changes from day to night
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'