Mechanistic studies on. beta. -ketoacyl thiolase from Zoogloea ramigera: Identification of the active-site nucleophile as Cys sub 89 , its mutation to Ser sub 89 , and kinetic and thermodynamic characterization of wild-type and mutant enzymes

Journal Article · Tue Jul 11 04:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00440a006· OSTI ID:5036731

Thompson, S; Mayerl, F; Walsh, C T ^[1]; Peoples, O P; Masamune, S; Sinskey, A J ^[2]

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1. Harvard Medical School, Boston, MA (USA)
2. Massachusetts Institute of Technology, Cambridge (USA)

Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys{sub 89} by acetylation with ({sup 14}C)acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t{sub {1/2}} of 2 min at pH 7, 25{degree}C. Cys{sub 89} has been converted to the alternate nucleophile Ser{sub 89} by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser{sub 89} enzyme retains 1% of the V{sub max} of the Cys{sub 89} enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the V{sub max} in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with ({sup 14}C)acetyl-CoA and isolation of the labeled Ser{sub 89}-containing tryptic peptide. Comparisons of the Cys{sub 89} and Ser{sub 89} enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of ({sup 32}P)CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA.

• OSTI ID: 5036731
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:14; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETYLATION
ACYLATION
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CHEMICAL REACTIONS
COENZYMES
DAYS LIVING RADIOISOTOPES
DOSE-RESPONSE RELATIONSHIPS
ENZYMES
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MICROORGANISMS
MUTANTS
NUCLEI
ODD-ODD NUCLEI
OXIDOREDUCTASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOISOTOPES
REACTION KINETICS