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Biosynthetic thiolase from Zoogloea ramigera. Evidence for a mechanism involving Cys-378 as the active site base

Journal Article · · Journal of Biological Chemistry; (USA)
OSTI ID:5768255
; ; ; ; ; ; ;  [1]
  1. Massachusetts Inst. of Tech., Cambridge (USA)
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Biosynthetic thiolase from Zoogloea ramigera was inactivated with a mechanism-based inactivator, 3-pentynoyl-S-pantetheine-11-pivalate (3-pentynoyl-SPP) where K1 = 1.25 mM and kinact = 0.26 min-1, 2,3-pentadienoyl-SPP obtained from nonenzymatic rearrangement of 3-pentynoyl-SPP where K1 = 1.54 mM and kinact = 1.9 min-1 and an affinity labeling reagent, acryl-SPP. The results obtained with the alkynoyl and allenoyl inactivators are taken as evidence that thiolase from Z. ramigera is able to catalyze proton abstraction uncoupled from carbon-carbon bond formation. The inactivator, 3-pentynoyl-SPP and the affinity labeling reagent, acryl-SPP, trap the same active site cysteine residue, Cys-378. To assess if Cys-378 is the active site residue involved in deprotonation of the second molecule of acetyl-CoA, a Gly-378 mutant enzyme was studied. In the thiolysis direction the Gly-378 mutant was more than 50,000-fold slower than wild type and over 100,000-fold slower in the condensation direction. However, the mutant enzyme was still capable of forming the acetyl-enzyme intermediate and incorporated 0.81 equivalents of {sup 14}C-label after incubation with ({sup 14}C)Ac-CoA for 60 min. The reversible exchange of {sup 32}P-label from ({sup 32}P)CoASH into Ac-CoA, catalyzed by the Gly-378 mutant enzyme, proceeded with a Vmax (exchange) 8,000-fold less than the wild type enzyme but at least 10-fold faster than the overall condensation reaction. These data provide evidence that Cys-378 is the active site base.
OSTI ID:
5768255
Journal Information:
Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 266:13; ISSN JBCHA; ISSN 0021-9258
Country of Publication:
United States
Language:
English

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59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
AMINO ACIDS
BACTERIA
BARYONS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
CARBON 14 COMPOUNDS
CARBON COMPOUNDS
CARBOXYLIC ACIDS
CATALYSIS
CHEMISTRY
CYSTEINE
DAYS LIVING RADIOISOTOPES
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
INACTIVATION
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
MEMBRANE PROTEINS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PROTEINS
PROTONS
PSEUDOMONAS
RADIOISOTOPES
REAGENTS
RECEPTORS
THIOLS
TRACER TECHNIQUES
TRANSFERASES