Single turnover kinetics of tryptophan hydroxylase: evidence for a new intermediate in the reaction of the aromatic amino acid hydroxylases.
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 I, 4.4 s(-1), matches that for formation of the 4a-hydroxypterin product monitored at 248 nm. Chemical-quench 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.
Pavon
Jorge Alex
JA
Department of Biochemistry and Biophysics, Texas A&M University, College Station,Texas 77843-2128, USA.
Eser
Bekir
B
Huynh
Michaela T
MT
Fitzpatrick
Paul F
PF
D024322:Amino Acids, Aromatic / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D013379:Substrate Specificity; D014365:Tryptophan Hydroxylase / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D024322:Amino Acids, Aromatic; D014443:Tyrosine; D010649:Phenylalanine; D006899:Mixed Function Oxygenases; D014365:Tryptophan Hydroxylase