105] Isopropylmalate isomerase (yeast)

Abstract

Publisher Summary This chapter presents the assay, purification, and properties of isopropylmalate isomerase from yeast. The enzyme is assayed by measuring either the increase or the decrease in optical density at 235 m μ due to the formation of dimethylcitraconate (DMC) from β -isopropylmalate ( β -IPM) or due to the disappearance of DMC. One unit of isomerase activity is the amount of enzyme that catalyzes the disappearance of 1 micromole of dimethylcitraconate per minute at 32° under the assay conditions. Specific activity is expressed as units per milligram of protein. The crystallized enzyme is specific for its substrates, β -IPM, α -IPM, or DMC and shows a linear relationship between enzyme activity and enzyme amount over a range of 20-fold. The enzyme is inhibited by the sulfhydryl compounds cysteine, glutathione, and mercaptoethanol. The optimal pH for the enzyme is 7.0. The crystallized enzyme also shows a single band in disc electrophoresis on acrylamide gel and a single, symmetrical peak in sedimentation velocity centrifugation experiments.