Purification and Properties of Methylisocitrate Lyase, a Key Enzyme in Propionate Metabolism, from Candida lipolytica

Abstract

Methylisocitrate lyase catalyzing the cleavage of threo-D_s-2-methylisocitrate into pyruvate and succinate was purified about 60-fold from cell free extracts of Candida lipolytica. The purified enzyme required a divalent cation and a sulfhydryl compound for maximum activity. Sulfhydryl reagents strongly inhibited enzyme activity. The molecular weight was estimated to be about 1.2×10⁵ by gel filtration. The enzyme was specific for threo-D_s-2-methylisocitrate (Km=7.7×10^-4M) and did not catalyze the cleavage of threo-DL-isocitrate. Attempts to detect condensation between pyruvate and succinate by the preparation were unsuccessful. The enzyme was activated by NAD but noncompetitively inhibited by NADH and NADPH: these results support the idea that this enzyme is also a regulatory enzyme of the methylcitric acid cycle concerning the oxidation of propionate to pyruvate.