Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes.
Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys89 by acetylation with [14C]acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t 1/2 of 2 min at pH 7, 25 degrees C. Cys89 has been converted to the alternate nucleophile Ser89 by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser89 enzyme retains 1% of the Vmax of the Cys89 enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the Vmax in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with [14C]acetyl-CoA and isolation of the labeled Ser89-containing tryptic peptide. Comparisons of the Cys89 and Ser89 enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of [32P]CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
S
S
Thompson
F
F
Mayerl
O P
OP
Peoples
S
S
Masamune
A J
AJ
Sinskey
C T
CT
Walsh
D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D009154:Mutation; D013816:Thermodynamics; D015045:Zoogloea / Q000201:enzymology*
D000217:Acyltransferases; D000102:Acetyl-CoA C-Acyltransferase