THE PURIFICATION, PROPERTIES AND INTERNAL PEPTIDE SEQUENCES OF ALCOHOL ACETYLTRANSFERASE ISOLATED FROM SACCHAROMYCES-CEREVISIAE KYOKAI NO 7

Alcohol acetyltransferase (AATase) catalyzes the esterification of isoamyl alcohol by acetyl coenzyme A. The enzyme was solubilized from the microsomal fraction of Saccharomyces cerevisiae Kyokai No. 7, using Triton X-100 and then purified by a series of chromatographic separations: Poly Buffer Exchanger 94 (PBE94), DEAE Toyopearl, Toyopearl HW60, hydroxyapatite, Octyl-Sepharose CL-4B, and hexanol-affinity column chromatography. When the solubilized enzyme was put on PBE94, two active fractions were obtained. The enzyme obtained after affinity column chromatography had a single band on an SDS polyacrylamide gel, and its molecular mass was estimated to be 60 kDa. The enzyme was most active at pH 8.0 and 25-degrees-C. It was stable between pH 7.5 and 8.5, but was unstable at tempratures above 10-degrees-C. The activity was markedly inhibited by heavy metal ions such as Cd2+, Cu2+, Zn2+, and Hg2+, and sulfhydryl reagents. The K(m) for acetyl-CoA was 0.19 mM. The internal peptide sequences were also identified.