The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[(2)H]indole.
The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan. Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[(2)H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4-2.0 in the reaction of 3-[(2)H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an alpha-subunit ligand, disodium alpha-glycerophosphate (Na(2)GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[(2)H]indole with tryptophan synthase preincubated with L-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the L-Trp external aldimine at 423 nm. The addition of Na(2)GP dramatically slows the rate of reaction of indole with the alpha-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[(2)H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na(2)GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na(+) or Na(2)GP. These results support the previously proposed mechanism for the beta-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.
Cash
Michael T
MT
Department of Chemistry, Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-2556, USA.
Miles
Edith W
EW
Phillips
Robert S
RS
D000179:Acrylates / Q000737:chemistry*; D001665:Binding Sites; D003903:Deuterium; D041961:Deuterium Exchange Measurement / Q000379:methods; D004789:Enzyme Activation; D017022:Flow Injection Analysis; D007211:Indoles / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry; D011485:Protein Binding; D021122:Protein Subunits; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D012486:Salmonella typhimurium / Q000201:enzymology*; D014367:Tryptophan Synthase / Q000737:chemistry*
D000179:Acrylates; D007211:Indoles; D009097:Multienzyme Complexes; D021122:Protein Subunits; D011732:Pyridoxal Phosphate; C030374:indole; D003903:Deuterium; D014367:Tryptophan Synthase