 |
|
Title
|
|
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.
|
|
|
Authors
|
|
S.Thompson,
F.Mayerl,
O.P.Peoples,
S.Masamune,
A.J.Sinskey,
C.T.Walsh.
|
|
|
Ref.
|
|
Biochemistry, 1989,
28,
5735-5742.
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
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.
|
|
|
|