BioChem 330 - Course Outline September

BioChem 330 - Course Outline • Metabolism and Bioenergetics (II) – ENZYME CATALYSIS: • kinetic constants kcat, Km • Catalytic strategies, the serine proteases – CATABOLISM (breakdown) • Carbohydrates – – – – Glycolysis Tricarboxylic Acid Cycle (TCA cycle 11.10.11) Electron Transport Chemiosmosis and ATPase • Fatty acids and amino acids Coenzyme A: Mobile Carrier for Acetyl Group from Pyruvate Acetyl Group C1 (terminal methyl) has oxidation state of -3 C2 (thioester) has oxidation state of +3 Rxns TCA cycle 1,2 Condensation and rearrangement 1. • • 2. 1. Citrate synthase: – - 8 kcal/mol 2. Aconitase: – + 3 kcal/mol • 3. 8. • – -8 kcal/mol 3,4 Oxidative decarboxylations • 4. 7. • • 6. 3. Isocitrate dehyd.: – - 5 kcal/mole 4. -ketoglutarate dehyd.: 5. 5,6,7,8 Rearrangements and regneration of OAA 5. Succinyl CoA synthetase: – -1 kcal/mole 6. Succinate dehyd: – 0 kcal/mol 7. Fumarase: – -1 kcal/mol • 8. Malate dehydro.: – +7.5 kcal/mol Reminder:Some bookkeeping for the TCA cycle • - The overall reaction Acetyl CoA + 3NAD+ + FAD + GDP + Pi + 2H20 2CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA 2 carbons in 2 carbons out Oxidn state Oxidn state -CH3 -3 CO2 2 x +4 -COS +3 -------------------------0 +8 Overall: 8 e- oxidation All e- leave in hydrogenations: FAD FADH2 (2 e-) 3NAD+  3NADH (3 x 2 e-) TCA1 Citrate Synthase two step reaction: condensation, hydrolysis [OAA ] = 1 uM limits the rate of reaction Product, citrate, is symmetric TCA1 Citrate Synthase OAA binds first, inducing conformational change in TCA1 dimer shown here TCA1 Mechanism for condensation Step 1 binding of OAA (-2 charge) facilitated through 2 His (+2 charge) D 375 abstracts H+ from -CH3 of acetylCoA (pKa ~18) to form enol intermed. nucleophilic attack of enol on keto of OAA to form citryl CoA complex Step 2 hydration of thioester to form citrate and regenerate acetylCoA TCA2 Aconitase Rearrangement Star represents the carbon derived from the -CH3 of acetyl CoA • despite symmetry, OH is ALWAYS moved away from • citrate is not chiral, yet enzyme can tell difference between the two identical -CH2COO- “arms” PROCHIRALITY: ProR and ProS “arms” of citrate. If starred carbon was C14, then citrate would be chiral, and this isomer would be proS. TCA2 Aconitase Structure Rearrangement facilitated by Fe4S4 cluster in center of TCA2 FeS cluster anchored to TCA2 by 3 cysteine bonds to Fe Citrate binds to Fe through both -OH and -COO on C3 Abstraction of a proton from * induces a cis aconitate intermediate, and conformational change as C goes from sp3 to sp2, then FeS cluster is in position to add (OH) to * position, away from C derived from acetyl CoA TCA3: the first oxidative decarboxylation Step 1 above is very similar to lactate dehydrogenase, oxidation of an alcohol to form a ketone, transfer of hydride to NAD+ Step 2 is loss of CO2 from central C, “enolate” intermediate (not shown), then protonation by solvent CO2 lost is NOT from original acetyl, but from COO- adjacent to ketone in OAA. TCA2 is a tetramer which shows positive cooperativity and product inhibition. TCA4: the second oxidative decarboxylation α-Ketoglutarate dehydrogenase complex is just like pyr dehyrdogenase complex This 2nd CO2 is also not from original acetyl CoA, but from COO- in OAA Process occurs through five steps: summary: decarboxylation by breakdown of TPP bound substrate, shuffling of atoms to mobile CoA and electrons to FAD by lipoyl arm, electrons then transferred from FADH2 to mobile carrier NAD+ TCA5: beginning of regeneration of OAA Breakdown of Acyl CoA coupled to substrate level phosphoryllation by succinate kinase (some isoenzymes of this use ADP rather than GDP). Hydrolysis itself is worth -6 kcal/mole, net DG is about 0 kcal because this energy used to phosphoryllate GDP TCA5: succinate thiokinase This O is from phosphate Step 1: Breakdown of Succinyl CoA by bound phosphate to form phosphate ester, free CoA released. Step 2:Breakdown of phosphate ester catalyzed by His, free succinate released and phosphoHis created. Step 3: Phosphohistidine phosphoryllates GDP, final conversion between GTP and ADP occurs on another enzyme assoc. (nucleotide diphosphate kinase). Phospho His swings out and over to nucleotide TCA5: Rossmann fold for nucleotide binding of purine portion of succinylCoA, ATP (or other nucleotide) grasp of β subunit for phosphoryllation TCA 6,7,8: regeneration of OAA TCA 6: succinate dehydrogenase enzyme has 1 FAD, 3 Fe-S centers and has a link to the ET chain, only trans isomer of fumarate is formed Redox potentials: fumarate + 2e-  succinate Eo’ = + 0.031 V FAD + 2e- FADH2 Eo’ = - 0.220 V Consider choice of FAD (-0.221 volts) vs NAD+ (Eo’ = -0.32 V) FAD, ΔEo’ = Eo FAD - Eo fumarate = -0.251 V ΔGo’ = -nF ΔEo’ = small positive NAD, ΔEo’ = Eo NAD - Eo fumarate = -0.351 V ΔGo’ = - nFΔEo’ = greater positive So choice of FAD keeps energy less positive TCA6,7,8: regeneration of OAA 7. 8. TCA7: fumarase hydrates fumarate to L-malate, in the process scrambling our labels from original acetyl group. Free energy less than zero. TCA8, malate dehydrogenase: third NADH generated, again, process akin to LDH, alpha keto acid generated by dehydrogenation and hydride shift to NADH. Free energy here large and positive, but drawn forward by exoergicity of next step. Note that neither of the carboxylic acids (the C which will be removed in the next cycle of TCA) has been derived from original acetylCoA. Tricarboxylic acid cylce  Highlights (reminder) of Redox chemistry A reminder about oxidation/reduction... e- • If net reaction is – X-  X + e – e- + H+  1/2 H2 • reference cell, H+, is reduced – H+ oxidizing agent, • sample cell, X-, is oxidized, – X- is reducing agent, • electrons flow from sample to reference cell, Sample cell Reference Cell – If X has a more negative reduc potential than ref, H+, spontaneous direction for electrons to flow is from X to H+ – CHECK: you’ll need to reverse X redu potential to reflect its oxidation, and add it to red potential of H+ to reflect net change in E – ΔE0’ is positive, rxn spontaneous – ΔG0’ = -nFΔE0’ < 0, rxn spontaneous For example... • So: NADH  NAD+ + H+ + 2eUbiQ + 2H+ + 2e-  UbiQH2 NADH + UbiQ + H+  NAD+ UbiQH2 Eo’ = +0.320 V Eo’ = +0.045 V ΔEo’ = + 0.365 V • And ΔGo’ = - nFΔEo’ = - (2)(23.1 kcal/V*mol)(.365 V) = -17 kcal/mole An overview of what happens to NADH... Electrons are transferred from NADH to O2 through a chain of three large protein complexes called NADH-Q reductase, cytochrome reductase, and cytochrome oxidase. Electron flow within these transmembrane complexes leads to the pumping of protons across the inner mitochondrial membrane [and thereby to the synthesis of ATP] • These are big • and These are little