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 temperatures above 10 degrees C. The activity was markedly inhibited by heavy metal ions such as Cd2+, Cu2+, Zn2+, and Hg2+, and sulfhydryl reagents. The Km for acetyl-CoA was 0.19 mM. The internal peptide sequences were also identified.
Minetoki
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General Research Laboratory, Ozeki Corp, Hyogo, Japan.
Bogaki
T
T
Iwamatsu
A
A
Fujii
T
T
Hamachi
M
M
D000123:Acetyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D005227:Fatty Acids; D008670:Metals; D011506:Proteins; D013439:Sulfhydryl Reagents; D000123:Acetyltransferases; C079238:alcohol O-acetyltransferase