J. Biol. Chem., Vol. 263, Issue 30, 15400-15406, Oct, 1988

Mutated forms of phosphoglycerate mutase in yeast affect reversal of metabolic flux. Effect of reversible and irreversible function of an enzyme on pathway reversal

N Gautan
Molecular Biology Unit, Tata Institute of Fundamental Research, Bombay, India.

Phosphoglycerate mutase (GPM) functions reversibly in the glycolytic pathway. Mutations altering the reversibility of GPM have been obtained in the yeast, Saccharomyces cerevisiae. While wild-type cells grow on glycolytic (glucose) or gluconeogenic (ethanol) substrates, cells with altered GPMs fall into three categories based on their phenotypes 1) glucose- ethanol-, 2) glucose+ ethanol-, and 3) glucose- ethanol+. Cells with the first two phenotypes possessed GPMs that functioned irreversibly in the glycolytic direction. Cells that were glucose- ethanol+ possessed an enzyme that functioned reversibly. All of the altered GPMs had maximal velocities that were less than 3% of the wild- type level. The properties of the altered GPMs studied here provide a rationale for the occurrence in the glycolytic pathway of several glycolytic enzymes such as GPM, which function at high velocities in relation to the much smaller metabolic flux that they support. The altered GPMs were purified and estimates of their kinetic constants obtained. Free energy profiles were drawn for catalysis by the wild type and a mutant GPM that functioned irreversibly. The mutant enzyme was very inefficient. It was shown that an enzyme that functions irreversibly at a reaction with a Keq value close to 1 would necessarily be inefficient while it could evolve to be efficient when catalyzing a reaction that has a Keq value much greater than 1. In the glycolytic path this could be the reason for the characteristic presence of enzymes that function irreversibly at reactions with large Keq values.
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