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

• Published in: FEMS microbiology letters Vol. 228; no. 1; pp. 111 - 113
• Main Authors: Bamforth, C.W, Kanauchi, M
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier B.V 07.11.2003; Blackwell Publishing Ltd; Blackwell
• Subjects: Acetyl Coenzyme A - metabolism; Acetyl-CoA; Alcohol acetyl transferase; Biological and medical sciences; Chemistry Techniques, Analytical - methods; Clinical Enzyme Tests; Coenzyme A - metabolism; Electrophoresis, Polyacrylamide Gel; Esters - analysis; Esters - metabolism; Ethanol; Ethanol - metabolism; Ethyl acetate; Fundamental and applied biological sciences. Psychology; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Indicators and Reagents; Methanethiol; Methyl thioacetate; Microbiology; Mycology; Nitroblue Tetrazolium; Saccharomyces cerevisiae - metabolism; Sulfhydryl Compounds - metabolism; Ethyl acetate; Alcohol acetyl transferase; Methyl thioacetate; Acetyl-CoA; Ethanol; Methanethiol; Yeast; Enzyme; Transferases
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/S0378-1097(03)00742-0

Ethanol- and methanethiol-dependent removal of acetyl-CoA by crude extracts of ale yeast has been monitored using a decrease in OD 232. 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°C and 60°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 K m for acetyl-CoA, suggesting that the alcohol or thiol substrate is the more likely substrate to be limiting.