Use of novel assays to indicate that O-esters and S-esters are produced by the same enzyme in brewing yeast

Ethanol- and methanethiol-dependent removal of acetyl-CoA by crude extracts of ate yeast has been monitored using a decrease in OD232. Activity has also been detected in these extracts after fractionation on polyacrylamide gels, in this case using a novel assay in which the coenzyme A produced in the reaction is linked via DCPIP reduction to color formation from nitroblue tetrazolium. Ethanol-and methanethiol-dependent activities migrate identically on such gels, and only one band of color formation was observed. Furthermore they displayed closely similar sensitivity to heating at 40degreesC and 60degreesC and pH optima, with activity maximal at pH 7.5. It is likely that a single enzyme is responsible for the formation of O-esters and S-esters in yeast. Initial kinetic studies indicate that methanethiol has higher affinity for the enzyme than has ethanol and a higher maximum velocity. However, the enzyme has a much lower K-m for acetyl-CoA, suggesting that the alcohol or thiol substrate is the more likely substrate to be limiting. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.