Purification and characterization of an acetyl-CoA hydrolase from Saccharomyces cerevisiae.
Acetyl-CoA hydrolase, which hydrolyzes acetyl-CoA to acetate and CoASH, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 1080-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, gel filtration and hydroxylapatite. The molecular mass of the native yeast acetyl-CoA hydrolase was estimated to be 64 +/- 5 kDa by gel-filtration chromatography. SDS/PAGE analysis revealed that the denatured molecular mass was 65 +/- 2 kDa and together with that for the native enzyme indicates that yeast acetyl-CoA hydrolase was monomeric. The enzyme had a pH optimum near 8.0 and its pI was approximately 5.8. Several acyl-CoA derivatives of varying chain length were tested as substrates for yeast acetyl-CoA hydrolase. Although acetyl-CoA hydrolase was relatively specific for acetyl-CoA, longer acyl-chain CoAs were also hydrolyzed and were capable of functioning as inhibitors during the hydrolysis of acetyl-CoA. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate, a histidine-modifying reagent.
Lee
F J
FJ
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Lin
L W
LW
Smith
J A
JA
D000104:Acetyl-CoA Hydrolase / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000302:isolation & purification*
D000596:Amino Acids; D006882:Hydroxyapatites; D017886:Durapatite; D013869:Thiolester Hydrolases; D000104:Acetyl-CoA Hydrolase