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 – Electron Transport – Chemiosmosis and ATPase • Fatty acids and amino acids BioChem 330 Nov 8, 2011
• Linkage of cytosolic events (glycolysis) to mitochondrial events (tricarboxylic acid cycle - TCA) in the diges on of carbohydrates • The linker: pyruvate dehydrogenase What’s happening to the substrate?
• ..in the pyruvate dehydrogenase complex
– Conversion of pyruvate to Acetyl CoA and CO2 • Unlike ac on of LDH, there is no regenera on of NAD+ for glycolysis • No going back • … then in the Krebs cycle
– Conver ng acetylCoA to 2 CO2 – The Cycle: origin and proper es A Bed me Story by PLW
Curly: Meets... Type: ? Mo Mo lity a specialty Eubacterium Mo is eaten by Curly!! Electron transport to
acceptor (O2)
1. Protozoa 2. Fungi 3. Plants 4. & Animals Curly/Mo Jrs I & II • Curly – Cytoplasm And now... – Glycolysis • Mo – Mitochondria...
..where, for our purposes... The Coupling Machinery is Complex • Pyruvate -> Acetyl CoA – (Oxida ve Decarboxyla on) - + – CH3-CO-CO2 + NAD + CoA-SH -> CH3-CO-S-CoA + NADH + CO2 – ΔGo’ = - 8 kcal/mole
• Catalyzed by pyruvate dehydrogenase complex
– E1 (24 α2β2 tetramers), E2 (8 α3 trimers ), and E3 (6 αβ dimers)
• “Stoichiometric” cofactors – NAD+ – CoA-SH • “Cataly c” cofactors – Thiamine pyrophosphate – Lipoic Acid (fa y acid) – FAD How it starts...
• First the E1 bound TPP (related to vitamin B1, absence of which causes beriberi) …does some carbanion chemistry on pyruvate...
… and now the “aldehyde” “Ac ve aldehyde” gets oxidized by running into oxidized lipoate on E2... …and then the drama c wrap-up is trapped by the CoA
This thioester
While the electrons from substrate stay with the lipoamide and ...
….go first to a bound FAD
….and finally end up in a NADH mobile e- carrier The components of which can be broken down into...
• THE ENZYMES: • E1: pyruvate dehydrogenase • E2: Dihydrolipoyl transacetylase • E3: Dihydrolipoyl dehydrogenase All of which occurs in a factory which is enormous... • Located in the matrix of the mitochondrion and larger than ribosomes Blueprint of the factory...
* E2’s assemble to form the octameric core : [(E2)3]8 * E1’s assemble in a dimeric way on each of the twelve edges of the cube
of E2 [(E1)2]12 *E3’s assemble in dimeric way on each of the six faces of E2 each:[(E3)2]6
•The 24 dehdrogenase proteins (E1) on Blow up of one E2 the outside of the complex bind pyruvate and release CO2 •The 24 transacetylases (E2) proteins shu le remaining atoms to CoA and the electrons to E3, •The 12 dehydrogenases E3 takes electrons from E2 through the FAD and ul mately to NAD+ To what end?
1. Ac ve aldehyde 2. Aceyllipoamide 3. Reduced/oxidized lipoamide 4. Reduced/oxidized FAD Rxns TCA cycle • 1. Citrate synthase: – - 8 kcal/mol • 2. Aconitase: – + 3 kcal/mol • 3. Isocitrate dehyd.: – - 5 kcal/mole • 4. α-ketoglutarate dehyd.: – -8 kcal/mol • 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 Some bookkeeping...
• The overall reaction - The net reaction of the cycle is
+ 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-) 3 NAD+ → 3NADH (3 x 2 e-) What is the significance of the “cycle”?
• All intermediates are “cofactors” in a broad sense – That fact was how the cycle was discovered (superstoichiometric s mula on of pyruvate oxida on by the various intermediates, and par cularly citrate, succinate, α-KG) • Pulled forward by hydrolysis of bound intermediate (citrylCoA) Biosynthe c considera ons... • Du es: Implica ons of those du es • They may betray the origins of the cycle in anaerobic bacteria Mitochondrial func on in normal and diabe c β-cells PIERRE MAECHLER AND CLAES B. WOLLHEIM
Figure 1 A map of human mitochondrial DNA indica ng diabetes-associated muta ons. The mito genome encodes 37 genes (16,569 bp): 13 polypep des, 22 tRNAs. 2 ribosomal RNAs The polypep des are cons tuents of the respiratory-chain complexes: 7 complex I subunits, 1 complex III subunits, 3 subunits of complex IV, 2 subunits of complex V The genes for tRNAs are presented as one-le er symbols. Muta ons in four of these tRNA genes are associated with diabetes: those for leucine (L), serine (S), lysine (K) and glutamic acid (E) tRNAs. Mitochondrial func on in normal and diabe c β-cells PIERRE MAECHLER AND CLAES B. WOLLHEIM
Figure 2 The TCA cycle and respiratory chain in a mitochondrion. Substrate oxida on in the TCA cycle ac vates the respiratory chain, leading to the genera on of ATP, which is subsequently translocated to the cytosol. ANT, adenine nucleo de translocator; GDH, glutamate dehydrogenase; Glu, glutamate; KG, ketoglutarate; OAA, oxaloacetate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; Suc-CoA, succinyl- CoA; SDH, succinate dehydrogenase; TCA, tricarboxylic acid; UCP2, uncoupling protein 2. Mitochondrial func on in normal and diabe c β-cells PIERRE MAECHLER AND CLAES B. WOLLHEIM Figure 3 Model for coupling of glucose metabolism to insulin secre on in the b-cell. Glucose is phosphorylated by glucokinase (GK) and converted to pyruvate (Pyr) by glycolysis. Pyruvate preferen ally enters the mitochondria and fuels the TCA cycle, resul ng in the transfer of reducing equivalents to the respiratory chain, leading to hyperpolariza on of the mitochondrial membrane ( m ) and genera on of ATP. Subsequently, closure of K ATP-channels depolarizes the cell membrane ( c ).
2+ 2+ 2+ This opens voltage-gated Ca channels, raising the cytosolic Ca concentra on ([Ca ]c), which triggers insulin exocytosis. Several puta ve messengers, or addi ve signals, proposed to par cipate in the metabolism–secre on coupling are indicated.