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The biosynthesis of methylcitrate.

S W Weidman, G R Drysdale

The Biochemical Journal

|January 1, 1979

Summary

Citrate synthase produces two methylcitrate isomers from propionyl-CoA and oxaloacetate, with isomer ratios affected by temperature. The enzyme also facilitates rapid proton exchange in propionyl-CoA, crucial for understanding metabolic disorders.

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Area of Science:

Biochemistry
Enzymology
Metabolic Disorders

Background:

Citrate synthase is a key enzyme in the citric acid cycle.
Methylcitrate is implicated in propionic acidemia, a metabolic disorder.
Understanding the stereochemical mechanisms of citrate synthase is crucial for metabolic research.

Purpose of the Study:

To investigate the stereochemical outcomes of methylcitrate synthesis by citrate synthase.
To elucidate the mechanism of proton exchange in propionyl-CoA catalyzed by citrate synthase.
To correlate enzyme activity with methylcitrate excretion in propionic acidemia.

Main Methods:

In vitro enzymatic assays using propionyl-CoA and oxaloacetate.
Temperature-dependent analysis of diastereoisomer formation.
Deuterium exchange experiments (2H2O) to study proton dynamics.
Kinetic analysis comparing proton exchange rates with Vmax.

Main Results:

Citrate synthase forms two methylcitrate diastereoisomers, with proportions sensitive to temperature.
The enzyme catalyzes rapid exchange of alpha-protons of propionyl-CoA with 2H2O in the presence of oxaloacetate and a thiol trap.
The pro-S proton exchange is significantly faster (15x) than the pro-R proton exchange.
Proton exchange rates exceed Vmax by over 1000-fold (pro-S) and 100-fold (pro-R).

Conclusions:

The stereoselective proton exchange mechanism of citrate synthase is demonstrated.
Findings provide insights into the formation of specific methylcitrate isomers observed in propionic acidemia.
The study highlights the enzyme's complex catalytic mechanism beyond simple condensation.

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