Week 1 AND 2 of Biochemistry Metabolism
Week 1 AND 2 of Biochemistry Metabolism BIOL 5311
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This 125 page Class Notes was uploaded by an elite notetaker on Saturday January 30, 2016. The Class Notes belongs to BIOL 5311 at Southern Methodist University taught by Dr. Vogel in Winter 2016. Since its upload, it has received 8 views. For similar materials see Biochemistry: Metabolism in Biology at Southern Methodist University.
Reviews for Week 1 AND 2 of Biochemistry Metabolism
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Biol/Chem5311 - Metabolism Pia Vogel office: 233, DLS lab: 221 phone: 214-768-1790 firstname.lastname@example.org Thermodynamics Background Thermodynamics Background Free energy changes are additive Thermodynamics Background Now some chemistry Standard Free Energy Change and Equilibrium of a Reaction • Nucleophiles, electrophiles • Carbon-carbon, carbon-hydrogen bond cleavage, formation • Carbonyl chemistry • Isomerization, rearrangements, elimination • Group transfer reactions • Redox reactions • ATP and other high energy compounds 1 Carbohydrate review Glycolysis • Chapter 7.1 and 7.2 (just glycogen) • Classification • Fischer convention • Epimers • Linear-cyclic form • Glycogen 3VuBHNlYwNzYwRjb2xvA2JmMQR2dGlkA1ZJUDUzMV8x?p=glycolysis+rap+songZ45XNyoA;_ylu=X3oDMTB0OWZjY LacticAcid Fermentation, short circuitingglycolysis Alcohol Fermentation through decarboxylation of pyruvate and reductionto alcohol Recyclingof NAD into glycolysis Gluconeogenesis Medicalrelevance 2 Regulationof metabolicpathways Pentose-phosphate pathway Citricacid cycle Glycogen Electron transport Pyruvate dehydrogenase 3 OxidativePhosphorylation– The ATPsynthase Fatty acids– β oxidation Adipose tissue Fatty acid synthesis Cholesterol - Steroid metabolism Amino acid degradation, Urea cycle Amino acid synthesis Porphyrin synthesis 4 Nucleotidesynthesis- degradation Putting it all together Excerpts from the syllabus Excerpts from the syllabus • GRADING: The course grades will be determinedaccording to the following scheme: Important Dates: • Your grade will reflect the average of 4 midterm exams and February9 EXAM 1 one finalexam. March3 EXAM 2 • The final exam is cumulative and willbe counted only to March6-13 Spring Break improve the grade. April 5 No class • Up to 10 % of the grade may be from quizzes April 6 Drop date • Grading scale: A: 93-100;A-: 90-92;B+:87-89;B: 83-86; April 7 EXAM 3 April 28 EXAM 4 and last class B-: 80-82;C+:77-79;C: 73-76;C-:70-72;D+: 67-69;D: 63-66; May 6 FINAL EXAM (Friday,8 a.m.) D-: 60-62;F:< 60 • Homework assignments will be available but will not be graded. Homework assignments andkeys willbe available on Blackboard 5 Carbohydrate review • Chapter 7.1 and 7.2 (just glycogen) • Classification • Fischer convention • Epimers • Linear-cyclic form • Glycogen Carbohydrates • Named so because many have formula C (H O) n 2 n • Produced from CO and2H O via2photosynthesis in plants • Range from as small as glyceraldehyde (M = 90wg/mol) to as large as amylopectin (M w 200,000,000 g/mol) • Fulfill a variety of functions including: – energy source and energy storage – structural component of cell walls and exoskeletons – informational molecules in cell-cell signaling • Often covalently linked with proteins to form glycoproteins and proteoglycans Classification • By carbonyl group: aldoses, ketoses • By number of carbon atoms: trioses, tetroses, pentoses, hexoses, heptoses • Combination of terms allows classification: aldopentose (e.g. ribose), ketopentose (e.g. ribulose), aldohexose (e.g. glucose) Aldoses and Ketoses An aldose contains an aldehyde functionality A ketose contains a ketone functionality Stereochemistry of carbohydrates, Fischer convention Enantiomers: stereoisomers that are non-superimposable mirror images The chiral center that is most distant from the carbonyl carbon is determines whether D or L. D and L isomers of a sugar are enantiomers Epimers, differ in the orientation at one of the chiral C-atoms Epimers of aldoses Epimers of ketoses Hemiacetals and Hemiketals • Aldehyde and ketone carbons are electrophilic • Alcohol oxygen atom is a nucleophilic • When aldehydes are attacked by alcohols, hemiacetals form • When ketones are attacked by alcohols, hemiketals form Saccharides form hemiacetals or hemiketals upon cyclization Cyclization of Monosaccharides • Pentoses and hexoses readily undergo intramolecular cyclization • The former carbonyl carbon becomes a new chiral center, called the anomeric carbon • The former carbonyl oxygen becomes a hydroxyl group; the position of this group determines if the anomer is α or β • If the hydroxyl group is on the opposite side (trans) of the ring as the CH2OH moiety the configuration is α • In the hydroxyl group is on the same side (cis) of the ring as the CH 2H moiety, the configuration is β Pyranoses The anomeric carbon is usually drawn on the right side Furanoses The anomeric carbon is drawn on the right side a- and b-forms of saccharides: here fructose, it can form both furanose and pyranose Chain-ring Equilibrium and Reducing Sugars • The ring forms exist in equilibrium with the open- chain forms • Aldehyde can reduce Cu 2+to Cu (Fehling’s test) • Aldehyde can reduce Ag to Ag (Tollens’ test) • Allows to detect reducing sugars, such as glucose Monosaccharides are reducing agents Complex carbohydrates by linkage of monosaccharides Common disaccharides milk sugar common house hold sugar Polysaccharides • Natural carbohydrates are usually found as polymers • These polysaccharides can be – homopolysaccharides – heteropolysaccharides • Polysaccharides do not have a defined molecular weight. – This is in contrast to proteins because unlike proteins, no template is used to make polysaccharides Glycogen • Glycogen is a branched homopolysaccharide of glucose – Glucose monomers form (a1 → 4) linked chains – Branch-points with (a1 → 6) linkers every 8-12 residues – Molecular weight reaches several millions – Functions as the main storage polysaccharide in animals Glycogen: a way to store glucose Carbohydrate Metabolism 1. Glycolysis, fermentation, glycogen Chapter 14.1 -14.3, pages 543-568, chapter 7.2, 255-256 https://video.search.yahoo.com/search/video;_ylt=A0LEV12MsLZUjiUAZ45XNyoA;_ylu=X3oDMTB0OWZjY 3VuBHNlYwNzYwRjb2xvA2JmMQR2dGlkA1ZJUDUzMV8x?p=glycolysis+rap+song Aldolase reaction 1406_class_I_aldolase.html Glyceraldehyde-3-P dehydrogenase Remember: Phosphoglycerate mutase Feeder molecules for Glycolysis: glycogen, starch, dissacharides, hexoses Lactic Acid Fermentation, short circuiting glycolysis The regenerated NAD is brought back in at the glyceraldehyde-3P step to allow synthesis of two moreATP without use of O . 2 Happens when O can2ot be brought to muscle fast enough. Lactate build up converted to glucose in gluconeogenesis usingATP generated when aerobic metabolism happens again. Alcohol Fermentation through decarboxylation of pyruvate and reduction to alcohol Recycling of NAD into glycolysis Pyruvate decarboxylase using TPP Alcohol dehydrogenase Medical Relevance High glycolysis rate in tumors • Druggable targets: inhibitors of glycolytic enzymes to starve the cancer cell of ATP: 2- deoxy glucose, lonidamine, 3-Br-pyruvate. Inhibit glycolysis and pentose phosphate pathway. • Diagnostics: PET scan, positron emission tomography uses 1F-2-deoxygucose enriched in cancerous tissue Defective glucose uptake in diabetes type 1: metabolic consequences Medical Defective glucose uptake Relevance in diabetes type 1: • Glucose metabolism limited by glucose uptake and phosphorylation by hexokinase • GLUT 1-3 always present in hepatocytes and brain neurons always expressed and present at membrane • GLUT 4 in skeletal, cardiac muscle and adipose tissue only upon insulin signaling • Without insulin (not produced in type 1 diabetes), high glucose in blood, while cells starve. Use fatty acids from fats and β- oxidation to make acetyl CoA to make ketone bodies as alternative fuel. Leads to ketoacidocis and low blood pH. Glucose: https://search.yahoo.com/search?p=glucose+song&ei=UTF-8&fr=chrf-yff34 Summary Glycolysis •Near universal pathway to oxidize glucose into 2 pyruvate, two ATP and two NADH •10 glycolytic enzymes in cytosol, 10 intermediates with either 6 or 3 C, all are phosphorylated •Preparatory phase: ATP invested to make 2 x C3-P molecules •Pay-off phase: both C3-P are converted into 2 pyruvate, thereby generating 2 ATP and 2 NADH/H + Feeder molecules for Glycolysis: glycogen, starch, dissacharides, hexoses Glycogen Chapter 7.2, 255-256 • Main storage polysaccharide in animals • α1→4 linked glucose with α1→6 linked branches. Extremely branched. • Especially abundant in hepatocytes • Form clusters of smaller granules of single, highly branched glycogen molecules with Mw=several Mio. • Each branch ends with non- reducing sugar • Glucose is removed from non- reducing ends • Multiple degradative enzymes can break down glycogen simultaneously → high speed • About 7% of liver wet weight, • Corresponds to about 0.4 M glucose! Blood [glucose] about 5 mM. Why is glycogen used as storage instead of glucose Breakdown of glycogen, Chapter 14.2, pp. 560 • Glycogen phosphorylase cleaves glycosidic bond making Glucose-1-P until branching point is reached • Debranching enzyme removed branches • Phosphoglucomutase catalyses reversible Glucose-1- P to Glucose-6-P • Glucose-6 P can then enter glycolysis • Net gain of one less ATP needed to form Glucose-6-P • However, for glycogen synthesis, glucose is activated with UTP, so same energy balance See later for regulation of glycogen synthesis-degradation Other monosaccharides that enter glycolysis Fructose or mannose Galactose (hydrolysis of lactose) •Fructose + ATP ↔ Fructose 6-P → glycolysis (in muscles and kidney) •Fructose + ATP ↔ Fructose 1-P (in liver) → dihydroxyacetone-P and glyceraldehyde which is then phosphorylated to GA-3-P using ATP. GA-3-P to enter glycolysis •Mannose + ATP → Mannose-6-P isomerized to Fru-6-P Feeder molecules for Glycolysis: glycogen, starch, dissacharides, hexoses Continuous throughput of energy in living systems Review Thermodynamics, reaction classes in Biochemistry Reading: pages 23-27, 192-198, Chapters 13.1, 13.2, 13.3 -1st law of thermodynamics: energy in a system is conserved -2 law of thermodynamics: the universe tends towards maximum disorder -free energy as indicator of „spontaneity“ (favored reaction) -chemical equilibrium -enzyme catalyzed reactions, reaction velocity -common reaction types Organisms transform energy and matter from their surroundings Thermodynamics tells us which of the reactions are possible and happen spontaneously and which reactions need to be fueled by other reactions. Thermodynamics in Biochemistry • First law of thermodynamics: Energy (U) in a system is conserved ΔU=q-w, energy change=absorbed heat – work performed Enthalpy (H)= Energy that includes pressure and volume of a system (usually pressure is constant) H= U+pV ΔH= ΔU + p ΔV Thermodynamics in Biochemistry • Second law of thermodynamics: Entropy of the universe (S) increases Free Energy (G) indicates spontaneous reaction • G= H-TS • ΔG= ΔH - T ΔS G= amount of energy capable of performing work during a reaction at constant T and P; H = heat content of a reaction (what kinds of chemical bonds, interactions, etc); ΔH negative for exothermic reactions, positive for endothermic reactions S = quantitative expression for degree of randomness, more or less molecules formed, higher/lower organization of molecules, ΔS positive for higher randomness A reaction is spontaneous – favored - when ΔG<0 Entropy: state of disorder Are reactions favorable? A reaction is favorable when ΔG<0 Free energy changes are additive Standard Free Energy Change and Equilibrium of a Reaction Conventions in Biochemistry o Standard state as defined in physical chemistry: 25 C (298 K), 1 atm pressure, pure elements in their most stable form (O , not O ), pH=0 2 3 Standard state in biochemistry: 25 C (298 K), 1 atm pressure, dilute solutions (1M), pH=7, if H O, H + 2+ 2 or Mg are parts of a a reaction, their concentrations are not included in equations. o ΔG = standard free energy change (under standard P-chem conditions) ΔG‘ =Standard transformed free energy change (under standard biochemical conditions) Both ΔG and ΔG‘ are physical constants for a given reaction Reactions at equilibrium aA + bB ⇔ cC + dD Actual free energy change of a reaction is a variable that depends on ∆G or ∆G‘ and the concentration of reactants. ∆G = ∆G + RT ln ([C] [D] / [A] [B] ) a b Actual ([C] [D] / [A] [B] ) of a reaction = Q=mass action ratio When ∆G < 0, reaction moves forward, when ∆G >0, reaction reverse, when ∆G =0, reaction at equilibrium o - ∆Go/RT ∆G = -RT ln k , oeq k = e eq At equilibrium: k = [C] [D] / [A] [B] =k /k b eq 1 2 or ∆G = -RT ln k‘ eq Example problems: Calculate the equilibrium constants for the hydrolysis of the following o compounds at pH 7 and 25 C: a) Phosphoenolpyruvate: ∆G ’= -61.9 kJ mol -1 b) Pyrophosphate: ∆G ’= -33.5 kJ mol -1 Calculate the equilibrium constant for the reactionA + B ↔ C + D at 25 C. o The equilibrium concentrations are [A] = 10 µM, [B] = 15 µM, [C] = 3 µM and [D] = 5 µM. Is the reaction spontaneous? Consider a reaction with ∆ H = 15 kJ and ∆S = 50 J K . Is the reaction spontaneous at (a) 10 C or (b) 80 C o Calculate the values of ∆G’ at 25 C for the following reactions: a) C H6O 12lu6ose) ⇔ 2 (CH CH OH) + 2 CO3 2 2 b) C H6O 12lu6ose) ⇔ 2 (CH CHOHCOO ) (l3ctate) + 2 H - + The ∆G’ of (in kJ mol ) for glucose are -917.2, for ethanol –181.5, for lactate –516.6, and for CO -582.1 . Are the reactions spontaneous? Why? Thermodynamic background of enzyme activity Un-catalyzed reaction: ≠ How to Lower ∆G ? Enzymes organize reactive groups into proximity • Uncatalyzed bimolecular reactions: two free reactants → single restricted transition state conversion is entropically unfavorable • Uncatalyzed unimolecular reactions: flexible reactant → rigid transition state conversion is entropically unfavorable for flexible reactants •Catalyzed reactions: Enzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complex: Entropy cost is paid during binding reactant complex → transition state conversion is entropically OK Thermodynamic background of enzyme activity Enzymes stabilize the transition state of a reaction Equilibrium and Velocity (rate) of a reaction ∆G = ln keq, keq = e -∆G/RT keq will be large when ∆G is very negative, If keq is large, [Product] is large. Describes the equilibrium distribution of product and substrate, but not the rate of reaction. Rate constant for monomolecular reaction: V (velocity of reaction) = k [S], k is proportionality constant or rate constant. Rate constant for bimolecular reaction: V = k [S1] [S2], k takes into account the rate and probability of transition state formation and the energy of the transition state: k= KT/h x e -∆G#/R, ∆G = ∆G of transition state, or ∆G of activation. K = Boltzmann constant, h=Planck’s constant. The smaller ∆G , the faster the reaction! Now some chemistry • Nucleophiles, electrophiles • Carbon-carbon, carbon-hydrogen bond cleavage, formation • Carbonyl chemistry • Isomerization, rearrangements, elimination • Group transfer reactions • Redox reactions • ATP and other high energy compounds C-C and C-H bonds Carbonyl facilitate electron movement, imines are isoelectronic Some carbonyl reactions: formation, breaking of C-C bonds Example for C-C bond formation during cholesterol Biosynthesis, C+ is formed through excellent leaving group, PPi Isomerization reactions Elimination reaction: Free Radical Reactions: Free radical formation through Vitamin B 12, SAM, some reductase reactions, others Group transfer reactions: Phopshoryl transfer: Oxidation states of biochemical/organic compounds Oxidation of lactate to pyruvate Common Redox partners: NAD(P) /NAD(P)H H + Common Redox partner: Flavin nucleotides as stepwise e- acceptors Transition metals - the other redox partners: High energy compounds: the power ofATP Actual free energy change vs standard free energy change for ATP-hydrolysis Remember: ΔG and ΔG‘ are physical constants for a given reaction But: ∆G = ∆G + RT ln ([C] [D] / [A] [B] ) b Here: o ∆G = ∆G + RT ln ([ADP] [Pi] / [ATP] [H O]2 ∆G = ∆G + RT ln Q Depending on ATP/ADP ratio, the phosphorylation power (∆Gp) of ATP differs. The power of ATP • Not only the special chemical characteristics of ATP, but also the way different cells can maintain different concentrations of ATP changes its potency. Other molecules large free energies: Other molecules large free energies: Other molecules large free energies: Adding energy to a reaction: activating substrate through phosphorylation Thioesters as high energy intermediates: see glyceraldehyde-3- P-dehydrogenase How can enzymes be involved in chemical reactions? • Some amino acid side chains have pK close to physiological pH, they can act as acid or base in acid-base catalysis or as nucleophiles. Importantly: the pK of amino acids changes depending on polarity of environment. In non-polar environment: ▯ pK of amines get lower ▯ pK of acids get higher both getting closer to physiological pH • Amino acids often involved: His, Cys, Se-Cys, Glu, Asp, Lys, Tyr.
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