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

TitleBiosynthetic thiolase from Zoogloea ramigera. Evidence for a mechanism involving Cys-378 as the active site base.
Publication TypeJournal Article
Year of Publication1991
AuthorsPalmer, MA, Differding, E, Gamboni, R, Williams, SF, Peoples, OP, Walsh, CT, Sinskey, AJ, Masamune, S
JournalJ Biol Chem
Volume266
Issue13
Pagination8369-75
Date Published1991 May 05
ISSN0021-9258
KeywordsAcetyl-CoA C-Acetyltransferase, Acrylates, Affinity Labels, Alkynes, Amino Acid Sequence, Binding Sites, Catalysis, Cysteine, Kinetics, Molecular Sequence Data, Mutation, Pantetheine, Protons, Sequence Homology, Nucleic Acid, Substrate Specificity, Zoogloea
Abstract

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 14C-label after incubation with [14C]Ac-CoA for 60 min. The reversible exchange of 32P-label from [32P]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.

Alternate JournalJ Biol Chem
Citation Key217
PubMed ID1673680