Single Turnover Kinetics of Tryptophan Hydroxylase: Evidence for a New Intermediate in the Reaction of the Aromatic Amino Acid Hydroxylases

摘要

Tryptophan hydroxylase (TrpH) uses a non- heme mononuclear iron center to catalyze the tetrahydropterin- dependent hydroxylation of tryptophan to 5-hydroxytryptophan. The reactions of the TrpH · Fe(II), TrpH · Fe(II)· tryptophan, TrpH · Fe(II)· 6MePH_4. tryptophan, and TrpH · Fe(II)· 6MePH_4· phenylalanine complexes with O_2 were monitored by stopped-flow absorbance spectroscopy and rapid quench methods. The second-order rate constant for the oxidation of TrpH · Fe(II) has a value of 104 M~(-1) s~(-1) irrespective of the presence of tryptophan. Stopped-flow absorbance analyses of the reaction of the TrpH · Fe(II)·6MePH_4·tryptophan complex with oxygen are consistent with the initial step being reversible binding of oxygen, followed by the formation with a rate constant of 65 s~(-1) of an intermediate I that has maximal absorbance at 420 nm. The rate constant for decay of 1, 4.4 s~(-1), matches that for formation of the 4a-hydroxypterin product monitored at 248 nm. Chemicalquench analyses show that 5-hydroxytryptophan forms with a rate constant of 1.3 s~(-1) and that overall turnover is limited by a subsequent slow step, presumably product release, with a rate constant of 0.2 s~(-1). All of the data with tryptophan as substrate can be described by a five-step mechanism. In contrast, with phenylalanine as substrate, the reaction can be described by three steps: a second-order reaction with oxygen to form I, decay of I as tyrosine forms, and slow product release.