BIOCHEMISTRY: TCA CYCLE, GLUCONEOGENESIS, ENZYME KINETICS

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Biochemistry II Lecture Notes Mark Mattie, MD, Ph.D.

TCA Cycle

Definition: The citric acid cycle (CAC) – also known as the TCA cycle (tricarboxylic acid cycle) or the Krebs cycle is a series of chemical reactions used by all aerobic organisms to release stored energy through the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins, into adenosine triphosphate (ATP) and carbon dioxide. In addition, the cycle provides precursors of certain amino acids, as well as the reducing agent NADH, that are used in numerous other reactions.

· TCA cycle overview

· Cycle provides energy in the form of reduced coenzymes for the production of ATP

· Nearly all CO2 produced by body is byproduct of TCA cycle and pyruvate dehydrogenase

· TCA cycle operates exclusively in mitochondria in proximity to e- transport chain and is considered to be part of the aerobic respiration pathway

· The cycle is a convergence pathway for oxidative metabolism of carbohydrates, AA’s, FA’s

· Carbon in molecules oxidized (burned) to CO2

· Removes e- from fat, carbohydrate, and protein to produce reduced coenzymes

· Coenzymes are then oxidized by e- transport chain to produce ATP

· TCA cycle can operate in two modes: catabolic and anabolic and is, therefore, described as being amphibolic

· Energy production (Catabolic mode)

· Carbohydrate, FAs, AA’s converted to acetyl-CoA

· Acetyl-CoA oxidized to produce:

· Reduced coenzymes by four redox reactions/cycle

· Three mitochondrial NADH molecules

· One mitochondrial FADH2 molecule

· One GTP by substrate level phosphorylation

· Biosynthesis (Anabolic mode)

· All biosynthetic reactions require input of carbons other than those contained in acetyl-CoA

· Input pathways are called anapleurotic (“filling up”) reactions

· TCA cycle intermediates are used for biosynthesis of:

· Glucose during starvation

· C’s taken from AA’s and lactate

· The conversion of carbohydrates to fat

· Nonessential AA’s

· E.g., Aspartate, glutamate

· Pyruvate is a substrate for many reactions in metabolism

· Pyruvate -> Lactate by lactate dehydrogenase

· Pyruvate -> Alanine by alanine aminotransferase (ALT)

· Pyruvate -> Oxaloacetate by pyruvate carboxylase

· Pyruvate carboxylase is homotetramer

· Carboxylases generally use CO2 and coenzyme biotin (aqueous vitamin) and ATP to drive carboxylation

· Each subunit contains allosteric binding site for acetyl-CoA

· Acetyl-CoA is a positive allosteric effector

· Acetyl-CoA produce by lipolysis during starvation will trigger production of oxaloacetate for gluconeogenesis

· Pyruvate -> Acetyl-CoA by pyruvate dehydrogenase complex

· Acetyl-CoA is a negative allosteric effector

· Pyruvate -> Acetyl-CoA catalyzed by pyruvate dehydrogenase complex

· Pyruvate dehydrogenase

· Catalyzes oxidative decarboxylation of pyruvate

· Multienzyme complex

· One of several α-ketoacid dehydrogenases

· Enzyme activity regulation

· Regulated by allosterism

· Negative allosteric effectors are Acetyl-CoA and NADH

· Regulated by and covalent modification

· Sequence of conversion

· Pyruvate transported from cytoplasm into mitosol via pyruvate-specific transporter

· Pyruvate converted to acetyl-CoA by pyruvate dehydrogenase complex located in the mitochondria

· Irreversible reaction

· Major source of acetyl-CoA for TCA cycle

· Multienzyme complex composition

· Functional Components

· (E1) Pyruvate dehydrogenase

· Coenzymes

· Thiamine pyrophosphate (TPP)

· Vit B1

· (E2) Dihydrolipoyl transacetylase

· Transfers acetyl group

· Transfers reducing equivalents to FAD

· Coenzymes

· Lipoic acid

· Coenzyme A

· (E3) Dihydrolipoyl dehydrogenase

· Transfers reduction equivalents to NAD+

· Coenzymes

· FAD

· NAD+

· Regulation of complex

· Activate and deactivate E1 of pyruvate dehydrogenase

· Protein kinase

· Kinase phosphorylates and inhibits E1

· Kinase allosterically activated by high energy state compounds

· ATP

· Acetyl-CoA

· NADH

· Kinase allosterically inhibited by low energy state compounds:

· NAD+

· Coenzyme A

· Kinase inhibited by pyruvate

· Feedforward mechanism

· Phosphoprotein phosphatase

· Dephosphorylation action activates E1

· Phosphatase activated by Ca2+ in skeletal muscle

· In skeletal muscle, Ca2+ promotes activity of phosphatase -> produces energy by activating E1

· Other regulation methods

· Induction and repression of protein synthesis

· Proteolysis of enzyme proteins

· Pyruvate Dehydrogenase deficiency

· Most common enzymatic cause of congenital lactic acidosis

· Unable to convert pyruvate to acetyl-CoA

· Equilibrium shifted in favor of converting pyruvate to lactic acid by lactate dehydrogenase

· TCA cycle reactions

0. Acetyl group from acetyl-CoA condenses with oxaloacetate

· Acetyl-CoA may originate from

· Carbohydrates

· Oxidative decarboxylation of pyruvate

· Triacylglycerol

· Oxidation of glycerol & FFAs

· FA’s bypasses pyruvate formation

· Proteins/AA’s

1. Synthesis of citrate from acetyl-CoA and oxaloacetate

· Catalyzed by citrate synthase

· Aldol condensation reaction forming citrate

· Synthase allosterically regulated

· Activated by Ca2+, ADP

· Inhibited by ATP, NADH, succinyl-CoA, fatty acyl-CoA derivatives

· Primary mode of regulation

· Availability of substrates acetyl-CoA and oxaloacetate

2. Isomerization of citrate to isocitrate

· Catalyzed by aconitase

· Inhibited by fluoroacetate (rat poison)

· Converted to fluoroacetyl-CoA

· Condenses with oxaloacetate to form fluorocitrate

· Fluorocitrate is an inhibitor of aconitase

· Citrate accumulation

3. Oxidation and decarboxylation of isocitrate to α-Ketoglutarate

· Catalyzed by isocitrate dehydrogenase

· Allosterically activated by ADP, NAD+ and Ca2+

· Increase in energy demand results in accumulation of ADP and NAD which stimulate isocitrate dehydrogenase

· Allosterically inhibited by ATP and NADH

· Citrate accumulates in mitochondria after carbohydrate rich meal

· Exported to cytosol for lipogenesis

· FA exported from liver for storage in adipose tissue as triglycerides

· Citrate allosteric inhibitor of PFK-1 and activates acetyl-CoA carboxylase

· Irreversible oxidative decarboxylation of isocitrate

· Yields first NADH of three by the TCA cycle

· Releases CO2

· One of TCA cycle rate-limiting steps

4. Oxidative decarboxylation of α-ketoglutarate to succinyl-CoA

· Catalyzed by α-ketoglutarate dehydrogenase complex

· Oxidative decarboxylation of ketoglutarate

· Releases 2nd CO2 produces 2nd NADH

· Inhibited by ATP, GTP, NADH, succinyl-CoA

· Activated by Ca2+

· Not regulated by Phosphorylation/Dephosphorylation

· Coenzymes required, same as pyruvate dehydrogenase

· Thiamine pyrophosphate

· Lipoic acid

· FAD

· NAD+

· Coenzyme A

· At this point in TCA cycle, the two C injected have been balanced by two removed in the form of CO2

· In two C in 2Acteyl-CoA -> out two C in 2CO2

· No net biosynthesis of intermediates when C sourced from acetyl-CoA

5. Cleavage of succinyl-CoA

· Catalyzed by succinate thiokinase (AKA succinyl-CoA synthetase)

· Cleaves high-energy thioester bond of succinyl-CoA

· Coupled to phosphorylation of GDP -> GTP

· Substrate level phosphorylation

· ATP and GTP are energetically interconvertible by nucleoside diphosphate kinase

· GTP + ADP <-> GDP + ATP

6. Oxidation of succinate to fumarate

· Catalyzed by succinate dehydrogenase

· Succinate dehydrogenase is also known as complex II of the e- chain

· Produces FADH2

· FAD -> FADH2

· FAD is the acceptor because reducing power of succinate insufficient to reduce NAD+

· FAD is the prosthetic group

7. Hydration of fumarate to malate

· Catalyzed by fumarase (fumarate hydratase)

8. Oxidation malate to oxaloacetate

· Catalyzed by malate dehydrogenase

· Produces 3rd and final NADH

· Energy produced by TCA cycle

· Two C enter cycle as acetyl group and converted to CO2

· As a result, there is no net production of TCA intermediates

· Total = 12 ATP/acetyl-CoA

· 11 ATP by oxidation of coenzymes

· Three mitochondrial NADH + H+ (production of 3 ATP)

· One mitochondrial FADH2 (production of 2 ATP)

· 1 ATP from GTP (via substrate level phosphorylation)

· Starting with one glucose molecule ending with e- transport chain results in the production of 36-38 ATP

· Variability result of use of malate vs. glycerol shuttle to transfer cytoplasmic redox equivalents of cytoplasmic NADH to mitochondrial matrix

1 Glucose Molecule

-------------------

Glycolysis

2 ATP -> 2 ATP

2 NADH cytosolic -> 2(2 to 3) ATP

Pyruvate DeH

2 NADH mitosolic -> 6 ATP

TCA

6 NADH mitosolic -> 18 ATP

2 FADH2 mitosolic -> 4

2 GTP -> 2 GTP

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Total: 36-38 ATP/glucose

· TCA cycle regulation

· Rate regulated points of the TCA cycle

· Citrate synthase

· Isocitrate dehydrogenase

· α-ketoglutarate dehydrogenase complex

· Availability of oxaloacetate

· As NADH & FADH2 accumulate, oxidized forms become depleted and the TCA cycle is inhibited due to depletion of oxidized coenzymes

· ADP

· Increases in ADP accelerates reactions generating ATP

· Oxidative phosphorylation stops if ADP is not available

· Oxidation and phosphorylation tightly coupled

· Anaplerotic (filling up) reactions

· Anaplerotic reactions provide source of C to produce a net increase in TCA intermediates

· Biosynthesis from TCA intermediates requires anaplerotic reactions supplying C, otherwise TCA cycle would stall when intermediates depleted

· E.g., Removal of succinyl-CoA for heme synthesis depletes mitochondrial oxaloacetate

· Acetyl-CoA does not provide any net increase in C for synthesis of TCA intermediates