Introduction to Biochemistry
Introduction to Biochemistry BIOCHEM 501
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Dr. Pablo Pollich
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This 9 page Class Notes was uploaded by Dr. Pablo Pollich on Thursday September 17, 2015. The Class Notes belongs to BIOCHEM 501 at University of Wisconsin - Madison taught by Samuel Butcher in Fall. Since its upload, it has received 21 views. For similar materials see /class/205190/biochem-501-university-of-wisconsin-madison in Biochemistry at University of Wisconsin - Madison.
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Date Created: 09/17/15
Biochem Part 1 main Points Lecture 2 0 Water 0 Hbonding potential 4 per H20 0 Average of 34 hbonds per h20 o Hydrophobic effect I Entropically unfavorable I Forces spontaneous clustering oflipids 0 Sum of all noncovalent interactions gives proteins their shape and structure Lecture 3 Proteins 0 Primary structure 0 Amino acids are L isomers o Nonpolar I Glycine I Alanine I Proline I Valine I Leucine I Isoleucine I Methionine 0 Polar I Serine I Threonine I Cysteine I Asparagine I Glutamine o Aromatic I Phenylalanine I Tyrosine I Tryptophan 0 Positive R groups I Lysine I Histadine I Arginine 0 Negative R groups I Aspartate I Glutamate lstpka 2 carboxylic acid 2ndpka 9 amino group pI isoelectric point pH where net charge is 0 pkapH when HA A peptide bond formation is a condensation reaction peptide bond breaking is a hydrolysis reaction Lecture 4 0 Protein purification o Solubilization disrupt call to isolate protein 0 Stabilization keep protein at 4 degrees C to keep stable 0 Detection 0 Light absorption 0 Tryptophan absorbs UV light around 280 nm I Most conjugated aromatic side chains 0 Electrophoresis I Separation on basis of charge I Can separate on basis of size when protein is denatured I PAGE smallest bands move faster larger bands move slower I Verify purity I Gives idea how big protein is 0 Purification 0 Ion exchange I Positively charged protein sticks to negatively charged resin bead I Eluted by changing pH or salt concentration 0 Gel Filtration Size exclusion I Separates based on size I smaller molecules move slower larger molecules move faster 0 Affinity Chromatography I Resin has a ligand bound to it the protein has a natural affinity for the ligand so it sticks to it o Isoelectric focusing I Purified by isoelectric point I Positive charge at the bottom negative charge at the top I Charged groups ionize and proteins travel down tube until they reach their pl 0 Sequencing 0 Proteins with same function as different organism have similar but not identical sequences 0 Differences arise from mutations 0 Provides a measure of evolutionary distance between species 0 Ifa protein sequence is conserved function ofproteins in any motion is disadvantageous for organism 0 Understanding basis ofmolecular disease 0 Not all amino acid changes are harmful 0 Overall strategy I Separation of charge ss reduction I AA composition analysis I N and C terminal analysis I Cleavage protein into fragments I Sequencing of fragments I Overlap of fragments I Identification of the location of disulfide bonds 0 Edmon Degradation I Hydrolysis of Nterminal residue I Remove 1 amino acid at a time 0 Mass spec I Can be used to sequence proteins 0 Tandem Mass Spec I Can be used to sequence protein by identifying fragments of unique mass Lecture 5 Myoglobin o 02 storage 0 Globular structure Peptide bond is planor due to partial double bond character 0 Small electric dipole between amide N and Carbonyl 0 Alpha helix 0 Most Right handed o 36 residues per turn each turn is 54 angstroms 0 side chains protrude every 100 degrees 0 can be many hundreds in length 0 R groups protrude outward Beta strands o Hbonds form between strands 0 Parallel and antiparallel 0 Side chains are on alternate sides of sheet 0 Few amino acid residues 310 0 Alpha carbons are 36 angstroms apart close enough to form Hbonds Fibrous proteins 0 Highly extended 0 Rep eating helical or Beta sheet structure 0 Abundant o Karatin 0 Highly tensile strength 0 Collagen I Triplehelical XRay diffraction crystallography o X ray hits molecule and diffracts 0 Electron density map is formed 0 Model building then structure is determined Forces that stabilize proteins 0 Hydrophobic effect 0 Ionic interactions Limit to Protein size 0 MW less than 100000 daltons o More e fficient to build large proteins from smaller structures 0 Error rate is 1 mistake per 1000 amino acids Affinsen s protein folding experiment 0 Denatured proteins 0 Removed denaturant and oxidized active protein 0 Oxidized to reform cross links while denaturant is present no active protein 0 Sequence determines structure Lecture 6 Protein Function 0 Heme o Porphyrin ring is at 0 Provides 4 nitrogen ligands to the iron 0 Helps stabilize Fe2 state Fe2 binds OZ Fe3 does not 0 When oxygen is bonded to Fe it Hbonds to a histidine 0 Carbon monoxide binds to free heme 20000 times stronger than Oxygen o Myoglobin discriminates bc od hbonding between oxygen and distal histidine o Myoglobin binding curve is hyperbolic o The lower the kd the tighter the oxygen binds o Myoglobin binds oxygen too tightly to be a transporter o Hemoglobin o 96 saturated in Lungs o 64 saturated in tissues 0 32 delivered to tissues 0 quaternary structure 2 alpha and 2 beta subunits 0 each subunit has a heme O o Rstate I I I I I o Tstate can bind up to 4 oxygen molecules Binds oxygen strongly Oxygen stabilized Favored at High pH When oxygen binds Fe is saturated and is symmetrically centered Fe tugs on helix F transmits conformational change More interactions between subunits Large hole in middle Predominant state ofhemoglobin at low oxygen levels Favored at low pH high C02 Stabilized by BPG BPG binds at positive residues in Tstate Fe is pulled closer to histadine Electrons are not centered on ring 0 When 02 binds Fe is shifted closer to histidine which changes conformation from T to R 02 binding is sigmoidal cooperative binding between 4 subunits Hemoglobin Transports C02 and H C02 transported by carbamylation of aminoterminal residue BPG regulated binding affinity of hemoglobin for 02 without it no opping RT Hemoglobin is allosteric protein multiple ligand binding sites binding at one site in uences binding at another I Sigmoidal curve 0000 O Allostery 0 binding ofligand at one site in uences binding at another site 0 sigmoidal kinetic behavior don t follow MM eqn Lecture 7 Enzymes Highly specific for substrates Reaction rate depends on substrate and activation barrier Enzymes lower activation energy and increase rate Enzymes complimentary to transition state Energy for lowering activation energy barrier acquired from favorable free energy of substrate binding to enzyme 3 catalytic strategies 0 General AcidBase catalysis I Provides functional group to aid in catalysis once enzyme is bound 0 Covalent catalysis I Formation of covalent bonds to enzyme 0 Metal ion catalysis I Stabilize charge build up at transition state Kinetics 0 Provides insight into chemical mechanism 0 ES to form EP is rat limiting 0 Rate of reaction proportional to ES 0 MM eqn I Assume early times in reaction product is negligible I Assume ES is constant 0 Km S at 12 VmaX 0 Km how tightly enzyme and substrate is bound 0 Kcat tells how quickly an enzyme can turn over I Catalytic activity Kcat Km Reversible inhibitors weak interactions 0 Competitive I will require more S to reach VmaX compete with S for active site I VmaX does not change 0 Uncomp etitive I Does not compete with substrate I Effects Km and Vmax equally 0 Mixed I Inhibitor binds to S and ES complex I Both Km and Vmax effected differently o Irreversible inhibition 0 Covalent modification 0 Inactivates enzymes Lecture 8 o Chymotrypsin Mechanism Substrate binds Side chain of residue to be cleaved nestles into hydrophobic pocket 0 Histidine and Serine interact to form alkoxide ion on serine his acts as base Ion attacks carbonyl carbon breaking double bond creating negative charge on oxygen Oxygen moves into oxyanion hole where it is stabilized by Hbonding to glycine and serine Instability of negative charge on oxygen leads to collapse of tetrahedral intermediate double bond reforms breaking peptide bond Amino leaving group is protonated by histadine his is general acid and leaves enzyme Water is deprotonated by histadine generating a hydroxide ion Hydroxide ion attacks carbonyl carbon breaking double bond generating second tetrahedral intermediate with oxygen in oxyanion hole stabilized by Hbonding with glycine and serine residues 0 Collapse of tetrahedral intermediate forms carbohydrate anion and displaces serine His and serine bound again 0 Diffusion second product from the active site regenerates free enzyme 0 Regulatory enzymes 0 Exhibit increased or decreased activity in response to certain signals 0 Allosteric enzymes I Bind regulatory compounds noncovalently I Have sigmoidal kinetic behavior I Positive modulators dial up activity I Negative modulators dial down activity 0 Covalently modified enzymes I Regulatory compounds are covalently attached in a reversible manner I Enzymes act on another enzyme to covalently modify it I Phosphorylation o Zymogens I Enzymes made as inactive precursors that need to be cleaved to become active 00 O O O OO 0 Lipids 0 Principal forms of energy stores 0 Fatty Acid I Building block of lipid I Polar head alphatic hydrophobic tail 0 Saturated lipid have higher melting points than unsaturated o Triacylglycerols I 3 Fatty acids attached to glycerol I Principle component ofadipose cells I Long term energy storage I 3 carbons 3 hydroxyl groups I highly reduced a lot of electrons I provide more than 2 times as much energy of carbohydrates I dehydrated take up less space I metabolized more slowly than glycogen starch or carbohydrates 0 Glycerophospholipids I Glycerol 3phosphate The backbone of glycerophospholipids I Glycerol 2 fatty acids phosphate group alcohol 0 Sphingolipids I Sphingosine backbone instead glycerol 3P I Immunogenic determinants in blood 0 Sterols Cholesterol major sterol in animals I Fused ring predominantly hydrophobic alkyl tail and polar head group I Steroid derived from cholesterol 0 Testosterone estradiol cortisol aldosterone prednisone I Vitamin D processed though UV light in skin 0 Fluid mosaic model I Membranes are dynamic structures but still have shape Micelles hydrophobic center polar head groups surrounding Bilayer polar head groups 0 outside and inside with hydrophobic layer in between 0 Liposome polar head groups on outside and in center lipid hydrophobic layer in between 0 Membrane proteins 0 Responsible for almost all biological activity associated with membranes 0 Peripheral membrane proteins I Associated with membrane I Dissociated by gentle means 0 Integral membrane proteins I Tightly associated with membrane I Require detergent for removal oflipid bilayer OO I Never invert I Inserted into the membrane in one orientation 0 Insoluble in water 0 Only extracted from lipid bilayers with detergents o Transmembrane alphahelacies I 2025 amino acid residues characteristic of trans membrane helix 0 Bacteriorhodopsin I Amino terminus outside carboxyl terminus inside I Light triggers conformational change drives protein transport 0 Beta Barrel I Hydrophobic residues face lipid bilayer I Hydrophilic residue line pore and upper and lower outer surfaces 0 Passive transporters I Selectivity for charge and size 0 Gated channels I Controlled by an external signal 0 Glucose Transporter I 4 major domains 12 transmembrane alpha helices I helices form hydrophilic channels through which glucose is transported I Conformational change occurs as glucose is transported from one site of membrane to the other 0 Active transport 0 Primary driven by ATP hydrolysis 0 Secondary driven by a gradient established by primary active transport 0 NAK pump I Primary active transport I Hydrolyses ATP and uses energy to pump ion out I Maintains low NA and high K concentrations I Pumps 3 NA ions out for every 2K ions pumped in I Generated membrane potential 0 Motor proteins 0 Myosin I Interacts with actin in muscle contraction I 2 supercolied Alpha helices I amino terminus 17 nm heads I 150nm tail with carboxyl terminus aggregates to form a thick filament Factin is a long filamentous polymer of Gactin subunits When muscle contracts I band narrows Z disk comes closer together Head groups interact with actin causing contraction Muscle contraction Conformational changes in myosin head are coupled to ATP hydrolysis ATP binds to myosin head causing dissociation from actin ATP is hydrolyzed causing a conformational change ADP and Pi remains associated with myosin head movement catalyzed by hydrolysis of ATP Myosin head attaches to actin filament causing release of Pi Phosphate Pi release triggers quotpower Stroke Conformational change in myosin head moves actin and myosin filaments relative to one another ADP is released
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