A kinetic study of homocitrate synthetase activity in the yeast Saccharomycopsis lipolytica

Abstract

1. 1.|A rapid method for estimating the activity of the first enzyme of lysine biosynthesis in yeasts (acetyl-coenzyme A: 2-ketoglutarate C-acetyl transferase, EC 4.1.3.21) is described. 2. 2.|In the wild type strain, the fixation of one substrate, S-acetyl coenzyme A, shows sigmoidal saturation kinetics. The initial rate experiments indicate that the reaction obeys an ordered mechanism, 2-ketoglutaric acid binding before S-acetyl coenzyme A. 3. 3.|The activity is completely inhibited in vitro by lysine and by some lysine analogs, which all show cooperative binding and have an heterotrophic effect on 2-ketoglutaric binding sites. A second class of effectors is found, including 2-aminoadipic acid, pipecolid acid and dipicolinic acid, which all affect the cooperativity of S-acetyl coenzyme A binding sites. 4. 4.|Two types of mutations which modify these inhibition patterns without affecting the catalytic activity are described. One results in a desensitization towards lysine and lysine analogs only. The other entirely abolishes the susceptibility towards the second type of inhibitors, without affecting the susceptibility to lysine. 5. 5.|No variations of the specific activity could be detected in the wild type strain at all; mutants showing an increased or a reduced activity were isolated. 6. 6.|Our results do not support the existence of isoenzymes at the level of homocitrate synthetase in this yeast.