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

摘要: 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 nonenzymatic rearrangement of 3-pentynoyl-SPP 1.54 1.9 min-1 an affinity labeling reagent, acryl-SPP. The results the alkynoyl allenoyl inactivators are taken as evidence that Z. is able to catalyze proton abstraction uncoupled carbon-carbon bond formation. acryl-SPP, trap same active site cysteine residue, Cys-378. To assess if Cys-378 residue involved in deprotonation second molecule acetyl-CoA, Gly-378 mutant enzyme studied. In thiolysis direction more than 50,000-fold slower wild type over 100,000-fold condensation direction. However, still capable forming acetyl-enzyme intermediate incorporated 0.81 equivalents 14C-label after incubation [14C]Ac-CoA for 60 min. reversible exchange 32P-label [32P]CoASH into Ac-CoA, catalyzed by enzyme, proceeded Vmax (exchange) 8,000-fold less but at least 10-fold faster overall reaction. These data provide base.