α-Isopropylmalate Synthase from Yeast: Purification, Kinetic Studies, and Effect of Ligands on Stability

Author:

Ulm E. H.1,Böhme R.1,Kohlhaw G.1

Affiliation:

1. Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

Abstract

α-Isopropylmalate synthase, the first specific enzyme in leucine biosynthesis, was purified approximately 100-fold from extracts of Saccharomyces sp. (strain 60615), the most effective step being specific elution with the feedback inhibitor leucine from a hydroxyapatite column. In the early steps of purification, special care was taken to protect the synthase against proteolytic activities. The apparent molecular weight of the enzyme as determined from gel filtration on a calibrated column was 137,000 in the absence and 121,000 in the presence of leucine. Inhibition by leucine was specific and strongly p H-dependent, with the leucine concentration necessary for half-maximal inhibition increasing about 10-fold as the p H increased from 7.5 to 8.5. Within this p H range, catalytic activity remained almost unchanged. The apparent K m values for the two substrates were found to be 16 μ m for α-ketoisovalerate and 9 μ m for acetyl-coenzyme A. K + was required for full activity, the apparent K a value being 2 m m . Leucine inhibition was of the mixed type, resulting in decreased V max and increased apparent K m values forboth substrates. Whereas no cooperative effects were observed with either substrate, positive cooperativity was seen with leucine in the presence of saturating substrate concentrations. Leucine and, to a lesser extent, α-ketoisovalerate stabilized the purified enzyme against heat-inactivation. The presence of acetyl-coenzyme A, on the other hand, accelerated the inactivation. In subsequent experiments, coenzyme A was recognized as the actual inactivating ligand, being effective even at lower temperatures and in concentrations which were estimated to be in the range of the enzyme concentration.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

Reference23 articles.

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