Glycogen(n) Glycogen(n-1) UDP-Glucose Glycogen(n-1) G-1-P ...kitto.cm.utexas.edu/.../Lecture2009/395GLec21_09.pdf · Citric Acid Cycle (chapter 21) - Review Fates of Pyruvate - TCA

G-1-P

Glycogen(n)

UDP-GlucoseGlycogen(n-1)

Glycogen(n-1)

UTPPP 2P

Other tissues, like the heart, may alter this pattern.

Citric Acid Cycle (chapter 21)- Review Fates of Pyruvate

- TCA Cycle Overview

- Source of Acetyl~SCoA

- Pyruvate Dehydrogenase Complex (PDC)

- Reactions / Structure / RegulationEnzymes of the Citric Acid Cycle

- Reactions / Energy Summary / Amphibolic Nature

Glyoxylate Cycle (chapter 23-2)- Glyoxysome / Mitochondrion Enzymes

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C6H12O6 + 6 O2 6 CO2 + 6 H2O ∆Go’ = -2823 kJ/mol

C6H12O6 2 C3H6O3 ∆Go’ = -196 kJ/mol

Overview of the LINKING step and the TCA cycle. Details will follow.

The University of Texas has played a prominent role in the discovery of

vitamins in metabolism

Words Coined by Roger J. WilliamsPantothenic acid, 1933A B-vitamin. (Greek pantothen = “from everywhere”; now known to apply equally well to many other nutrients)Williams, R. J., Lyman, C. M., Goodyear, G. H., Truesdail, J. H. and Holaday, D. Pantothenic Acid, A Growth Determinant of Universal Biological Occurrence. J. American Chemical Society, 1933; 55:2912-27.

Folic acid, 1941A B-vitamin. (Latin folium = leaf)Mitchell, H. K., Snell, E. E. and Williams, R J. The Concentration of “Folic Acid.” J. American Chemical Society,1941; 63:2284.

Avidin, 1941A protein in raw egg white that avidly binds biotin (a B-vitamin), making it unavailable. Eakin, R. E., Snell, E. E. and Williams, R. J. The Concentration and Assay of Avidin, the Injury-Producing Protein in Raw Egg White. J. Biological Chemistry, 1941; 140:535-43

Two of the three forms of vitamin B6, lipoic acid, avidin, folinic acid, synthesis of vitamin B12, and pioneering work on inositol.Roger J. Williams

(1893-1988)

William Shive

(1916-2001)

University of Texas

Biochemical Institute- vitamin discovery

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Lester J. Reed

(UT 1948-1997)

E1(TPP) E2(Lipoic Acid) E3(FAD)

Eli Lilly Award - 1958Merck Award - 1994

Pyruvate HSCoA NAD+

NADHCO2 Acetyl~SCoA

Figure 21-6 The five reactions of the PDC

Mitochondria: Site of the linking step and the TCA cycle

This organelle has an oxidizing environment, and interesting evolutionary history

The “linking step”: PDCBe sure to take your vitamins: Five cofactors are used in the PDC

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Figure 21-3a: Electron micrographs of the E. coli pyruvate dehydrogenase multienzyme complex. (a) The intact complex, (b) dihydrolipoyltransacetylase (E2) “core”.

Five Reactions of the PDC

Overall linking step reaction is misleadingly simple:

Pyr + NAD+ + CoA AcCoA + NADH + CO2

E1 uses a TPP cofactor. In PDC the hydroxyethyl TPP is not released as an aldehyde, as in pyr decarboxylase, but passed to lipoic acid on E2.

Figure 17-27Reaction mechanism of pyruvate decarboxylase.

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Figure 21-8 Domain structure of the dihydrolipoyltransacetylase (E2) subunit of the PDC.

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E2

E2 is the PDC core enzyme; it spontaneously assembles. In bacteria it forms a trimer and sits on the 3-fold of the aggregate structure.

Figure 21-12a: X-Ray structure of E1 from P. putidabranched-chain a-keto acid dehydrogenase. A surface diagram of the active site region shows TPP in a deep cleft that can be reached by the mobile E2 arm system.

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TPP

E1

Lipoyllysine arm is very mobile

Ac-lipoamide reaches active site with CoA

Reduced lipoic acid is re-oxidized by E3

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E3

Figure 21-13a: X-Ray structure of dihydrolipoamidedehydrogenase (E3) from P. putida in complex with FAD and NAD+ shows physical arrangement of redox pair.

Figure 21-14Catalytic reaction

cycle of dihydrolipoyl

dehydrogenase.

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E3

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Figure 21-16The reaction transferring an electron pair from dihydrolipoyl dehydrogenase’s redox-active disulfide in its

reduced form to the enzyme’s bound flavin ring.

E3

Figure 21-11c: Electron microscopy–based images of the bovine kidney pyruvate dehydrogenase complex at ~35 Å resolution. (c) A cutaway diagram as in Part b but with E3 dimers (Fig. 21-13a) shown at 20 Åresolution (red) modeled into the pentagonal openings of the E2 core.

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Mammalian Complex:

60 E2 (52 kDa)

30 E1(α2β2 154 kDa)

12 E3 dimers (110 kDa)

+ ~6 binding proteins

+ ~3 kinase (~62 kDa)

+ ~3 phosphatase (~100kDa)

PDC is regulated by products

High concentrations of NADH and/or AcCoAcan run reactions 3 & 5 backward

Figure 21-17b Factors controlling the activity of the PDC.(b) Covalent modification in the eukaryotic complex.

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On to the TCA cycle

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Hans Krebs

1937 C4C2

C6

C6

C4

C5C4

Simplified TCA Cycle

Figure 21-18a: Conformational changes in citrate synthase. (a) Space-filling drawing showing citrate synthase in the open conformation. (b) closed, substrate-binding conformation.

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TCA Cycle Enzymes

Figure 21-19 : Mechanism and stereochemistry of the citrate synthase reaction.

Aha! Enolate anion as a nucleophile.

Aconitase removes, and then adds

back, water

Aconitase has a 4 Fe-4S cluster. The FeS cluster carries out NO redox function, but interacts directly with an organic substrate.

In humans, a CYTOSOLIC form doubles as an iron monitor, regulating transcription from iron response elements (IRE).

Mechanism and stereochemistry of the aconitase reaction.Pa

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84

Figure 21-21Probable reaction mechanism of isocitrate

dehydrogenase.

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Oxidation creates a carbonyl electron sink to facilitate β-decarboxylation

The five reactions of the KGDC are similar to those of PDC,and the structure of KGDC is similar to the 24-mer PDC.

Figure 21-22a: Reactions catalyzed by succinyl-CoA synthetase. Formation of succinyl phosphate, a “high-energy” mixed anhydride.

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succinyl-CoA synthetase: the only direct phophorylation in TCA - step 1

Figure 21-22b: Reactions catalyzed by succinyl-CoA synthetase. Formation of phosphoryl–His, a “high-energy” intermediate.

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Figure 21-23Covalent attachment of FAD to a His residue of succinate dehydrogenase.

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Succinate dehydrogenase

- membrane bound enzymeFADH2

FAD

Standard Free Energy Changes (∆G°’) and Physiological Free Energy Changes (∆G) of TCA Cycle Reactions.

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Simplified TCA Cycle

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PDC

KGDC

ICDH

Cit Synth

Regulation of the citric acid cycle.

Flux through the system is largely controlled by concentrations of reactants and PRODUCTS, espNADH, at irreversible steps.

Another representation

of TCA regulation

, NADH

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Amphibolicfunctions of the citric acid cycle.

The intermediates of the TCA cycle are chemically very useful and are drawn off for a variety of tasks. Without CARRIERS, the cycle slows

Anaplerotic Reactions:

1) Pyruvate Carboxylase (has requirement for AcCoA– that makes sense!)

pyr + CO2 + ATP OAA + ADP

2) Malic E

pyr + CO2 + NADPH L-Mal + NADP+

3) Transamination Reactions:

Ala Pyr

Asp OAA

Glu KG

Figure 23-10:The glyoxylate cycle. Why plants can convert lipid to sugar and you can’t.

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The Glyoxylate shunt, found in plants and some microbes, allows synthesis of glucose from lipid derived AcCoA. Two novel enzymes short work with TCA to create the shunt.

Radioisotopes greatly facilitate metabolic mapping.

Putting label at differing places

allows researchers to follow enzyme activities and

construct pathways.

14C Dating14C is generated from N2 in atmosphere at ~constant rate. Living systems take it up, until they die. From then on, normal fraction of 14C decays with half live ~5700 years.