KREBS (= TCA) CYCLE

LEARNING OBJECTIVES for 2 Lectures

• Explain specific characteristics of Krebs cycle
• Explain the purpose of the cycle
• Define functions of the cycle
• Explain importance of pyruvate
• Explain specific characteristics of pyruvate carboxylase
• Explain specific characteristics of PDH enzyme complex
• Explain regulation of PDH complex
• Explain Leigh’s disease
• Define energy yield of the cycle
• Explain regulation of the cycle
• References Harper’s Illustrated Biochemistry and Lippincott Illustrated Reviews
Biochemistry, Google images, Medical Biochemistry Baynes JW, Dominiczak MH
 Krebs Cycle = Citric acid Cycle =
TCA (= Tri Carboxylic Acid Cycle)
• Located in Mitochondrion
• A shared pathway for the
metabolism of all fuels
• Oxidatively strips electrons from
Acetyl Coenzyme A
• Acetyl CoA is the common
product of catabolism of lipid,
carbohydrate, and proteins,
• Produce majority of Reduced
Coenzymes that are used for
ATP generation in ETC
• Although TCA cycle does not
use Oxygen in any of its
reactions
• Requires oxidative
metabolism in
Mitochondrion for
Reoxidation of reduced
coenzymes
• TCA cycle has 2 major
functions:
• Energy production and
Biosynthesis
 FUNCTIONS OF TCA CYCLE
• Acetyl-CoA is oxidized in TCA
cycle to produce;
• Reduced Coenzymes by 4 redox
reactions per turn of Cycle
• 3 produce Reduced Nicotinamide
Adenine Dinucleotide (NADH)
• 1 produces Reduced Flavin
Adenine Dinucleotide (FADH2)
• These reduced nucleotides
provide energy for ATP synthesis
by ETC
• One high-energy phosphate, Guanosine Tri Phosphate
(GTP), is also produced in the cycle by Substrate level
phosphorylation
• Nearly all Metabolic Carbon dioxide is produced by
Decarboxylation reactions catalyzed by
• Pyruvate dehydrogenase and TCA cycle enzymes in
Mitochondrion
 TCA Cycle
• In addition to its role in Catabolism;
• Participates in Synthesis of Glucose from Amino acids and
Lactate during Starvation and Fasting (Gluconeogenesis)
 TCA Cycle
• Participates in Conversion of Carbohydrates to Lipid
following a carbohydrate rich meal (Lipogenesis)
• Krebs cycle is a source of nonessential amino acids, such as
Aspartate and Glutamate
• Which are synthesized directly from Oxaloacetate and
α-ketoglutarate, respectively
• SuccinylCoA, which serves as a precursor to Porphyrins
for Heme synthesis
 TCA Cycle

• Begins with Acetyl CoA

• Has 3 major metabolic precursors
• In cytosol carbohydrates undergo Glycolysis to yield
Pyruvate which can be taken up by Mitochondria
• Pyruvate is oxidatively decarboxylated to Acetyl-
CoA by Pyruvate dehydrogenase enzyme complex
 During Lipolysis
• Triacylglycerols (= Triglycerides) are converted to
Glycerol and Free fatty acids,
• Which are taken up by cells and
• Transported into Mitochondria,
• Where they undergo Oxidation to Acetyl-CoA
 Proteolysis of Tissue Proteins
• Releases constituent Amino acids
• Many of which are metabolized to Acetyl-CoA and
TCA cycle intermediates
 Pyruvate carboxylase

• Uses CO2
• Catalyzes Carboxylation of Pyruvate to Oxaloacetate
• Coenzyme Biotin is covalently bound to enzyme
• Transfers Carbon originating from CO2 to Pyruvate
 Pryruvate is at a Crossroads in Metabolism
• May be converted in one step to;
• Lactate ( by Lactate dehydrogenase
= LDH), to Alanine (Alanine
aminotransferase= ALT), to
Oxaloacetate ( by Pyruvate
carboxylase), and to Acetyl-CoA
(by Pyruvate dehydrogenase
enyme complex)
• Depending on Metabolic
circumstances;
• Pyruvate may be routed toward
Gluconeogenesis , Fatty acid
synthesis , or TCA cycle
• Pyruvate is readily formed from
Lactate or Alanine
• Acetyl-CoA are derived from
Pyruvate through catalytic action of
Pyruvate dehydrogenase
• Oxaloacetate are derived from
Pyruvate through catalytic action of
Pyruvate carboxylase
 Pyruvate
• The end product of Aerobic Glycolysis
• Must be transported from Cytosol into Mitochondrion
• This is accomplished by a specific transporter
• That facilitates movement of Pyruvate across the inner
mitochondrial membrane
• Once in Mitochondrial matrix;
• Pyruvate is converted to Acetyl CoA by Pyruvate dehydrogenase
complex
• PDH complex is not part of the TCA cycle but
• Supplies substrate for TCA cycle
 Mechanism of action of
Pyruvate dehydrogenase complex

• TPP = Thiamine pyrophosphate; L = lipoic acid;

• CoA = Coenzyme A
 Components of PDH complex
• A protein aggregate of 3 Enzymes;
• Pyruvate decarboxylase (E1),
• Dihydrolipoyl transacetylase (E2), and
• Dihydrolipoyl dehydrogenase (E3)
• Each catalyzes a part of the overall reaction
• Their physical association links the reactions in proper
sequence without the release of intermediates
• PDH complex contains;
• 5 Coenzymes that act as carriers or oxidants for the
intermediates of the reactions
• E1 requires Thiamine pyrophosphate (TPP),
• E2 requires Lipoic acid and CoA, and
• E3 requires FAD and NAD+
• TPP, Lipoic acid, and FAD are tightly bound to the enzymes
and function as Coenzymes
 Mechanism of action of
Pyruvate dehydrogenase complex

• TPP = Thiamine pyrophosphate; L = lipoic acid;

• CoA = Coenzyme A
• TPP = Thiamine pyrophosphate; L = lipoic acid;
• CoA = Coenzyme A
 Mechanism of action of
Pyruvate dehydrogenase complex

• TPP = Thiamine pyrophosphate; L = lipoic acid;

• CoA = Coenzyme A
 Pyruvate dehydrogenase complex
• All Coenzymes of PDH complex, except for Lipoic
acid, are derived from Vitamins
• TPP is from Thiamine,
• FAD from Riboflavin,
• NAD from Niacin,
• CoA from Pantothenic acid
 Deficiencies of Thiamine or Niacin
• Can cause serious central nervous system problems
• If PDH complex is inactive; Brain cells are unable
to produce sufficient ATP via TCA cycle
• Wernicke-Korsakoff, an encephalopathy-psychosis
syndrome due to Thiamine deficiency, may be seen with
Alcohol abuse
• In addition to enzymes participating in conversion of
Pyruvate to Acetyl CoA,
• PDH complex also contains 2 tightly bound Regulatory
enzymes;
• Pyruvate dehydrogenase kinase (PDH kinase) and
• Pyruvate dehydrogenase phosphatase(PDH phosphatase)
 Regulation of
PDH enzyme complex
• Covalent modifications by 2 Regulatory enzymes that are
part of the complex alternately activate and inactivate
Pyruvate decarboxylase (E1)
• c-AMP independent PDH kinase; Phosphorylates and,
thereby, inactivates Pyruvate decarboxylase (E1)
• PDH phosphatase; Dephosphorylates and activates E1
• PDH kinase is allosterically
activated by ATP, Acetyl CoA,
and NADH
• Therefore, in the presence of
these high energy signals;
PDH complex is turned off
• So PDH complex becomes
inactive
• Pyruvate; a potent inhibitor of PDH kinase
• If Pyruvate concentrations are elevated,
• E1 will be maximally active
 CALCIUM
• A strong activator of
PDH phosphatase
• Stimulates E1 activity
• This is particularly important
in Skeletal muscles,
• Where release of Ca2+ during
contraction stimulates;
• PDH complex and, thereby,
energy production
• Although Covalent regulation by Kinase and Phosphatase is
primary,
• PDH complex is also subject to Product (NADH and Acetyl
CoA) inhibition
 A case
• A 7 months old child showed Progressive neurologic
deterioration characterized by
• Loss of coordination and muscle tone
• Unable to keep his head upright
• Great difficulty moving is limbs, which were limp
• Suffered from Acidosis
• Administration of Thiamine had no effect
• Lab analysis results; Blood levels of Lactate,
α-ketoglutarate, and Branched chain amino acids
 A case
• Child died a week later
• Liver, brain, kidney, skeletal muscle, and heart were examined
postmortem
• All Gluconeogenic enzymes were shown to have normal
activities
• Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase
were deficient
• Defective component was shown to be Dihydrolipoyl
dehydrogenase (E3)
• E3 is a single gene component required by all of the α-ketoacid
dehydrogenases
DEFICIENCIES IN PYRUVATE METABOLISM IN THE
TCA CYCLE
‘’ Leigh’s disease’’
• A group of disorders
• All characterized by Lactic acidosis
• Lactic acid accumulates under anaerobic conditions or
• Because of any enzyme defect in the pathway from Pyruvate to
ATP synthesis
• In this case;
• There are defects in both Pyruvat dehydrogenase and
α-ketoglutarate complexes as well as other
• α-keto acid dehydrogenase complexes required for the
Catabolism of Branched chain amino acids
 Leigh’s disease
• Failure of Aerobic metabolism leads to
• Increases in Blood levels of Lactate, α-ketoglutarate, and
Branched chain amino acids
• Tissues dependent on Aerobic metabolism such as brain
and muscle, are most severely affected
• Clinical picture includes;
• Impaired motor function,
• Neurologic disorders, and
• Mental retardation
 Leigh’s diesase
• Subacute necrotizing encephalomyelopathy
• A rare, progressive, neurodegenerative disorder
• Caused by defects in Mitochondrial ATP production,
• Primarily as a result of mutations in genes that code for
proteins of PDH complex, ETC, or ATP synthase
• Both nuclear and mitochondrial DNA can be affected
• 2 Carbon atoms enter
the cycle as Acetyl CoA
• Leave as 2 CO2
• Cycle does not involve;
Net consumption or
Production of
Oxaloacetic acid
• 4 pairs of electrons are
transferred during one
turn of the cycle
• 3 pairs of electrons
reducing 3 NAD+ to
NADH and
• 1 pair reducing FAD to
FADH2
 ENERGY PRODUCED BY KREBS CYCLE
Oxidation of 1 NADH by ETC leads to formation of 2.5 ATP
Oxidation of 1 FADH2 yields 1.5 ATP
Total yield of ATP from Oxidation of 1 molecule Acetyl CoA (Using both
Substrate level and Oxidative phosphorylation);
3 NADH ’den ; 7.5 ATP
1 FADH2 ’den ; 1.5 ATP
1 GTP ’den ; 1 ATP
Totally; 10 molecules of ATP molecules produced from
the oxidation of one molecule of acetyl coenzyme A
using both substrate-level phosphorylation and
oxidative phosphorylation.
 ATP Formation in the Catabolism of Glucose

TCA Cycle
Totally; 32 molecules of ATP molecules
produced from the oxidation of one molecule of
GLUCOSE using both substrate-level
phosphorylation and oxidative phosphorylation.
 REGULATION OF KREBS CYCLE
• In contrast to Glycolysis, which
is regulated primarily by PFK-1
• TCA cycle is controlled by the
regulation of several enzymes
• The most important of
regulated enzymes of TCA cycle
• Citrate synthase
• Isocitrate dehydrogenase
• α-ketoglutarate dehydrogenase
complex
• Reducing equivalents needed for oxidative
phosphorylation are generated by the Pyruvate
dehydrogenase enzyme complex and the TCA cycle.
• Both processes are upregulated in response to a decrease
in the ATP/ADP ratio