Distinction between isocitrate lyase and methylisocitrate lyase in Candida lipolytica.

Abstract

Examination of DEAE-cellulose chromatography clearly demonstrated the presence of the enzyme cleaving 2-methylisocitrate into pyruvate plus succinate in the methylcitric acid cycle. The enzyme differed from the usual isocitrate lyase of Candida lipolytica, and was herein given the trivial name of methylisocitrate lyase. The chromatography also proved the mutant strain of C. lipolytica, producing 2-methylisocitrate from odd-carbon n-alkanes, to lack this methylisocitrate lyase. The presence of isozyme of isocitrate lyase was not detected in C. lipolytica. The isocitrate lyase of C. lipolytica could cleave 2-methylisocitrate at a rate of 3% of that of isocitrate cleavage. On the contrary, methylisocitrate lyase hardly acted on isocitrate. Two lyases differed in chromatographic elution patterns, pH optima, kinetics of thermal inactivation, and behavior with inhibitors, such as itaconate and oxalate, in addition to substrate specificity. The experimental results revealed that methylisocitrate lyase is impossible to be the isozyme of isocitrate lyase, and justified the previously proposed ideas that propionyl-CoA is oxidized through the methylcitric acid cycle in C. lipolytica and that the absence of this enzyme in the mutant strain caused the extracellular accumulation of 2-methylisocitrate from odd-carbon n-alkanes.