CITRIC ACID CYCLE

-Anaplerosis
Reading:
Harpers Biochemistry Chapter 18
Lehninger Principles of Biochemistry
3rd Ed. pp. 584-592

OBJECTIVES

To understand the citric acid cycle as both a source

and sink for carbon compounds involved in other
metabolic pathways.
To understand anaplerosis as a mechanism for
regulating the flow of intermediates in the citric acid
cycle.
To understand how the citric acid cycle is essential
for supplying acetyl-CoA to the cytoplasm for use in
fatty acid biosynthesis.

In aerobic organisms, the citric acid cycle is an amphibolic pathway, one

that serves in both catabolic and anabolic processes.
Besides its role in the oxidative catabolism of carbohydrates, fatty acids,
and amino acids, the cycle provides precursors for many biosynthetic
pathways, through reactions that served the same purpose in anaerobic
ancestors.
Ketoglutarate and oxaloacetate can serves as precursors for glutamate
and aspartate, respectively, by simple transamination, which themselves
can act as precursors for other amino acids and nucleotides.
Oxaloacetate can be converted to glucose in gluconeogenesis.
Succinyl-CoA is a central intermediate of heme groups.

Role of the citric acid

cycle in anabolism.
Intermediates of the citric
acid cycle are drawn off
as precursors in many
biosynthetic pathways.
Shown in red are four
anaplerotic reactions that
replenish depleted cycle
intermediates

Anaplerotic reactions replenish

intermediates in citric acid cycle

As intermediates are removed to serve as biosynthetic precursors, they are replenished by anaplerotic reactions.
Under normal circumstances, removal and replenishment are in dynamic balance so intermediates stay almost
constant.

Most significant is the formation of

oxaloacetate by pyruvate carboxylase
ATP + CO2 + H2O + pyruvateoxaloacetate + ADP + Pi

Pyruvate carboxylase is a regulatory enzyme and is virtually inactive in the

absence of acetyl-CoA, its positive allosteric activator. Whenever acetyl-CoA, the
fuel for the citric acid cycle, is in excess, it stimulates the pyruvate carboxylase
reaction to make more oxaloacetate, enabling the cycle to proceed.
Pyruvate carboxylase has biotin as a prosthetic group. It is a specialized carrier
of one carbon groups in their most oxidized form, CO 2.
Biotin is required in the human diet, it is abundant in many foods and made by
intestinal bacteria. Biotin deficiency is rare but can happen when large quantities
of raw eggs are consumed as avidin in egg white is a tight biotin binder.

Regulation of the Citric Acid Cycle

Regulation of acetyl-CoA production by the

pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex of vertebrates is strongly

inhibited by ATP, by acetyl-CoA, and by NADH, the products of the
reaction catalyzed by the complex.
When long chain fatty acids are available, and can provide acetylCoA via B-oxidation, pyruvate oxidation is inhibited.
When too little acetate flows through the cycle, AMP, CoA, and
NAD+ all accumulate and allosterically activate the pyruvate
dehydrogenase complex.
Pyruvate dehydrogenase is inhibited by reversible serine
phosphorylation - the kinase responsible is allosterically activated
by ATP.

Regulation of the citric acid cycle at its

three exergonic steps

The flow of metabolites through the citric acid cycle is under stringent regulation.
Three major factors govern the rate of flux: substrate availability, inhibition by
accumulating products, and allosteric feedback inhibition of the enzymes that catalyze
early steps in the cycle.
Each of the three strongly exergonic steps - those catalyzed by citrate synthesis,
isocitrate dehydrogenase, and -ketoglutarate dehydrogenase can become the ratelimiting step under specific conditions:
-citrate synthase can limit the rate of citrate formation if substrates (acetyl-CoA and
oxaloacetate) are at low level
-high NADH/NAD+ ratio inhibits both dehydrogenase reactions
-product accumulation inhibits all three steps
-In muscle Ca2+, the signal for contraction and increased energy demand, activates
isocitrate, -ketoglutarate, and pyruvate dehydrogenases

The citric acid cycle takes part in fatty acid

biosynthesis

Acetyl-CoA is a major building block for long-chain fatty acid

synthesis (in non-ruminants; in ruminants, acetyl-CoA is derived from
acetate).
Since pyruvate dehydrogenase is a mitochondrial enzyme and the
enzymes needed for fatty acid biosynthesis are extramitrochondrial,
the acetyl-CoA is recovered as citrate, cleaved back to acetyl-CoA in
the cytosol by ATP-citrate lyase

The glycoxylate cycle in plants

Vertebrates cannot
convert fatty acids or
acetate to carbohydrates
In many organisms other
than vertebrates, the
glyoxylate cycle serves as
a mechanism for
converting acetate to
carbohydrate

Summary

The citric acid cycle is the final pathway for the oxidation of
carbohydrate, lipid, and protein. It catalyzes the combination of their
common metabolite, acetyl-CoA, with oxaloacetate to form citrate.
Through a series of dehydrogenations and decarboxylations, citrate is
degraded, producing reducing equivalents in the form of NADH and
FADH2, releasing CO2, and regenerating oxaloacetate.

The overall rate of the citric acid cycle is controlled by the rate of
conversion of pyruvate to acetyl-CoA and by the flux through citrate
synthase, isocitrate dehydrogenase, and -ketoglutarate
dehydrogenase. These fluxes are largely determined by the
concentrations of substrates and products; the end products ATP and
NADH are inhibitory.

Summary

Citric acid cycle intermediates are also used as precursors in

the biosynthesis of amino acids and other biomolecules. These
intermediates are replenished by anaplerotic reactions
catalyzed by pyruvate decarboxylase, PEP carboxykinase, PEP
carboxylase, and malic enzyme
The glyoxylate cycle in plants and some microorganisms
bypasses the two decarboxylation steps of the citric acid cycle
and makes possible the net formation of succinate and
oxaloacetate from acetyl-CoA, glucose formation from fatty
acids or acetate.