Alpha-Isopropylmalate synthase from Alcaligenes eutrophus H 16 I. Purification and general properties

Abstract

alpha-Isopropylmalate (IPM) synthase, the first enzyme in the biosynthesis of L-leucine, was purified to a specific activity of 12 micronmole/min x mg protein from the valine-isoleucine double auxotrophic mutant A-81 of the hydrogen bacterium Alcaligenes eutrophus H16. The activity in crude extracts of derepressed cells was 0.106 micronmoles of isopropylmalate formed per min and per mg protein. Gel electrophoresis and regel electrophoresis of the isolated main band resulted in several distinct bands, which were not altered by the additions of substrate alpha-ketoisovalerate, feedback inhibitor leucine or other effectors. The isoelectric points of the enzyme protein was between 3.9 and 4.0. The molecular weight was 114 500 daltons and 100 000 respectively in the absence and presence of the feedback inhibitor leucine. The enzyme activity depended strongly on the pH, the optimum is at pH 8.2. The enzyme was could labile and exhibits temperature anomalies.