The Citrate Cleavage Enzyme: III. CITRYL COENZYME A AS A SUBSTRATE AND THE STEREOSPECIFICITY OF THE ENZYME

Abstract

0.15 pmole; malate dehydrogenase, 0.2 unit;2 citryl-CoA, ap- proximately 0.1 pmole; citrate cleavage enzyme; and water in a final volume of 1 ml. The production of oxaloacetate in the presence of malate dehydrogenase caused an oxidation of NADH and a decrease in absorption at 340 mp. Absorbance readings were taken every 4 minute, and the rate of NADH oxidation from 1 to 3 minutes was used to calculate the rate of the cleavage reaction. Total amounts of citryl-CoA were measured in a similar system except that only 0.01 to 0.03 pmole of citryl-CoA was added to an excess of citrate cleavage enzyme, and the total decrease in absorbance at 340 rnp was determined. Sulfhydryl was measured according to the method of Ellman (8). Acetyl-CoA was determined enzymatically with citrate- condensing enzyme (9). Hydroxamate was measured by the method of Lipmann and Tuttle (10). Oxaloacetate was deter- mined enzymatically by using malate dehydrogenase and NADH and by measuring the total decrease in absorbance at 340 mp. Citryl-CoA was prepared by adding small aliquots of a tetra- hydrofuran solution of 0.25