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 tetra-nhydropterin-dependent hydroxylation of tryptophan to 5-hydroxytryptophan. The reactions of the TrpH3nFe(II),nTrpH3nFe(II) 3ntryptophan, TrpH3nFe(II) 3n6MePH4 3ntryptophan, and TrpH3nFe(II) 3n6MePH4 3nphenylalanine com-nplexes with O2 weremonitored by stopped-flow absorbance spectroscopy and rapid quenchmethods. The second-norder rate constant for the oxidation of TrpH3nFe(II) has a value of 104 M-1nsn-1nirrespective of the presence ofntryptophan. Stopped-flow absorbance analyses of the reaction of the TrpH3nFe(II) 3n6MePH4 3ntryptophan complexnwith oxygen are consistentwith the initial step being reversible binding of oxygen, followed by the formationwith anrate constant of 65 sn-1nof an intermediate I that has maximal absorbance at 420 nm. The rate constant for decaynof I, 4.4 sn-1n, matches that for formation of the 4a-hydroxypterin product monitored at 248 nm. Chemical-quenchnanalyses show that 5-hydroxytryptophan forms with a rate constant of 1.3 sn-1nand that overall turnover is limitednby a subsequent slow step, presumably product release, with a rate constant of 0.2 sn-1n. All of the data withntryptophan as substrate can be described by a five-step mechanism. In contrast, with phenylalanine as substrate,nthe reaction can be described by three steps: a second-orderreactionwithoxygentoformI,decayofIastyrosinenforms, and slow product release.