Krebs Cycle Enzymes

By Dr. Catherine Shaffer, Ph.D.Reviewed by Dr. Liji Thomas, MD

The Krebs cycle, also known as the citric acid cycle, is a series of enzymatic reactions that releases energy from stored carbohydrates, fats, and proteins.

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The Krebs Cycle was first proposed by Hans Adolf Krebs in 1937. The cycle also produces amino acid precursors and reduced nicotinamide adenine dinucleotide (NADH).  It is the primary metabolic pathway for all aerobic processes in animal tissue. In eukaryotes, the Krebs cycle takes place in the mitochondrial matrix whereas in prokaryotes, it occurs in the cytosol.

Krebs cycle steps

Acetyl-CoA is the starting point for the Krebs cycle. Throughout the eight reactions of the cycle, three molecules of NADH are produced and one of flavin adenine dinucleotide (FAD/FADH2). The following are the steps in the cycle:

1. Acetyl-CoA is combined with oxaloacetate by citrate synthase, to form a six-carbon molecule. Then the citric acid molecule is released from the enzyme complex.
2. A water molecule is removed from the 3’ position on citric acid and added back at the 4’ location by the enzyme aconitase resulting in isocitrate.
3. Isocitrate dehydrogenase catalyzes oxidation of a 4’ -OH group of isocitrate to yield alpha-ketoglutarate. One molecule of NAD is converted to NADH.
4. Alpha-ketoglutarate is decarboxylated, converting another molecule of NAD to NADH, by alpha-ketoglutarate dehydrogenase, yielding succinyl CoA which is an unstable compound.
5. Succinyl-CoA synthetase catalyzes the addition of a free phosphate group to guanosine diphosphate (GDP), creating guanosine triphosphate. In the process, the CoA group is released from succinyl-CoA. The resulting molecule is succinate.
6. Two hydrogens are released from succinate when succinate dehydrogenase reduces FAD to FADH2. The output of the reaction is fumarate.
7. Fumarase catalyzes addition of an -OH group to fumarate, producing L-malate.
8. In the final reaction of the cycle, oxaloacetate is regenerated by oxidation of L-malate by malate dehydrogenase. One molecule of NAD is converted to NADH.

Krebs cycle enzymes

The Krebs cycle enzymes are membrane proteins found within the matrix of the mitochondria except for succinate dehydrogenase which is an integral membrane protein locked to the inner mitochondrial membrane.

While NAD is the prosthetic group used to accept the protons generated during the three steps of oxidation, FAD is used by succinate dehydrogenase. The ultimate fate of these reduced coenzymes is to be reoxidized by entering the electron transport chain reactions in the inner mitochondrial membrane, when ATP is generated.

Some reactions of the Krebs cycle are close to thermodynamic equilibrium, and are, therefore, bidirectional. Those include enzymes interconverting succinate, fumarate, malate, and oxaloacetate. The reversibility of the reactions allows the generation of precursors for glucose synthesis, fatty acid and cholesterol synthesis, amino acid anabolism, nucleotides, and heme biosynthesis.

The Krebs cycle uses about two-thirds of the total oxygen consumed by the body, and generates about ⅔ of the energy. It plays a role in gluconeogenesis, transamination, deamination, and lipogenesis. Very few genetic abnormalities of the Krebs cycle have been found, possibly because it is critical for survival.

Evolution of the Krebs cycle

Most enzymes of the Krebs cycle are encoded in the nucleus in eukaryotes. It is believed that nuclear genes were acquired from ancestral mitochondrial genes during evolution. During that process, known as endosymbiosis, cells began living in symbiotic relationships.

The mitochondrion and chloroplast are supposed to have been originally free-living cells that eventually began living inside other cells. Prior to this event, Krebs cycle enzymes may have operated only as isolated steps in the host and mitochondria.

Different isoforms of the enzymes in different cell compartments may have arisen as a result of gene transfer events. The process by which these isolated steps in the cycle came together to form a complex, critical cycle necessary to life is not yet understood.

Further Reading

• All Biochemistry Content
• What is Biochemistry?
• What is Bioorganic Chemistry?
• An Introduction to Enzyme Kinetics
• How Can Biochemistry Practices be made Virtual?