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 ale 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 40 degrees C and 60 degrees C 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 Km for acetyl-CoA, suggesting that the alcohol or thiol substrate is the more likely substrate to be limiting.
Bamforth
C W
CW
Department of Food Science and Technology, University of California, Davis, CA 95616-8598, USA. cwbamforth@ucdavis.edu
Kanauchi
M
M
D000105:Acetyl Coenzyme A / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004796:Clinical Enzyme Tests; D003065:Coenzyme A / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters / Q000032:analysis* / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism; D007202:Indicators and Reagents; D009580:Nitroblue Tetrazolium; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D004952:Esters; D007202:Indicators and Reagents; D013438:Sulfhydryl Compounds; D009580:Nitroblue Tetrazolium; C005231:methylmercaptan; D000431:Ethanol; D000105:Acetyl Coenzyme A; D003065:Coenzyme A