Kinetic mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2 using the natural and alternative reactants.
The resonance-stabilized quinonoid 5-mercapto-2-nitrobenzoate (TNB) is a substrate for O-acetylserine sulfhydrylase-A (OASS-A) and -B (OASS-B), giving rise to the product S-(3-carboxy-4-nitrophenyl)-L-cysteine (S-CNP-cysteine) as confirmed by ultraviolet-visible and 1H NMR spectroscopies. A comparison of the kinetics of OASS-A and OASS-B indicates that the mechanism proceeds predominantly via a bi-bi ping pong kinetic mechanism as suggested by an initial velocity pattern consisting of parallel lines at low concentrations of reactants, but competitive inhibition by both substrates as the reactant concentrations are increased. Thus, in the first half-reaction, O-acetyl-L-serine (OAS) or beta-chloro-L-alanine (BCA) is converted to alpha-aminoacrylate in Schiff base with the active site pyridoxal 5'-phosphate, while in the second half-reaction cysteine (with sulfide as the reactant) or S-CNP-cysteine (with TNB as the reactant) is formed. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive. These data are consistent with acetate reversing the first half-reaction and producing more free enzyme to which acetate may also bind. Thus, there may be some randomness to the mechanism at high concentrations of the nucleophilic substrate.
Department of Microbiology, Texas College of Osteopathic Medicine, University of North Texas, Fort Worth 76107.
C H
CH
Tai
S R
SR
Nalabolu
T M
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Jacobson
D E
DE
Minter
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Cook
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003547:Cysteine Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D012486:Salmonella typhimurium / Q000201:enzymology*; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D013053:Spectrophotometry; D013861:Thiocyanates / Q000494:pharmacology; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000494:pharmacology
D007527:Isoenzymes; D013861:Thiocyanates; D015091:beta-Alanine; C006977:3-chloroalanine; D012694:Serine; D003547:Cysteine Synthase; C043943:O-acetylserine