Deuterium kinetic isotope effects for hydroxylation of the methyl group of 4-methylphenylalanine have been used as a probe of the relative reactivities of the hydroxylating intermediates in the aromatic amino acid hydroxylases phenylalanine, tyrosine, and tryptophan hydroxylase. When there are three deuterium atoms in the methyl group, all three enzymes exhibit an intrinsic isotope effect of about 13. The temperature dependence of the isotope effect is consistent with moderate tunneling, with the extent of tunneling identical for all three enzymes. In the case of phenylalanine hydroxylase, the presence of the regulatory domain has no effect on the values. The intrinsic primary and secondary isotope effects were determined using 4-methylphenylalanine containing one or two deuterium atoms in the methyl group. With one deuterium atom, the intrinsic primary and secondary effects have average values of 10 and 1.1, respectively. With two deuterium atoms, the primary effects decrease to 7.4 and the secondary effect increases to 1.3, consistent with coupled motion of the primary and secondary hydrogens. The results with all three enzymes are consistent with a hydrogen abstraction mechanism. The similarities of the isotope effects and extent of tunneling establish that the reactivities of the hydroxylating intermediates in the three enzymes are essentially identical.

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. ]-phenylalanine, are identical for Δ117PheH and Δ117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the D k cat value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6-to7-fold that for hydroxylation. In addition, there is a slight (∼34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With 2 H 5 -indole-tryptophan as a substrate for Δ117PheH, the D k cat value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the D k cat values are within error of those for Δ117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.Phenylalanine hydroxylase (PheH 1 ), tyrosine hydroxylase (TyrH), and tryptophan hydroxylase (TrpH) form a small family of nonheme iron monooxygenases (1). These enzymes catalyze

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.Tryptophan hydroxylase (TrpH)1 catalyzes the formation of 5-hydroxytryptophan (5-HOtrp) from tryptophan, the first and rate-limiting step in the biosynthesis of melatonin and serotonin (1,2). The enzyme belongs to the family of aromatic amino acid hydroxylases that also includes phenylalanine hydroxylase (PheH) and tyrosine hydroxylase (TyrH) (3). These three enzymes catalyze the hydroxylation of their corresponding substrates utilizing a tetrahydropterin and molecular oxygen (Scheme 1) (4-6). While the physiological reactions of PheH, TyrH, and TrpH are all aromatic hydroxylations, these enzymes will also catalyze benzylic and aliphatic hydroxylation (7-9). The eukaryotic forms of each enzyme are * Address correspondence to: Paul F. Fitzpatrick, Department of Biochemistry, MC 7760, University of Texas Health Science Center at San Antonio, San Antonio, fitzpatrick@biochem.uthscsa.edu,. † This work was supported by NIH grant R01 GM047291 and Welch Foundation Grant A1245 to PFF and NIH grant F31 GM077092 to JAP. 1 Abbreviations: TyrH, tyrosine hydroxylase; PheH, phenylalanine hydroxylase; TrpH, tryptophan hydroxylase; TauD, taurine:α-ketoglutarate dioxygenase; 6MePH 4 , 6-methyltetrahydropterin; 4a-HO-6MePH 3 , 4a-hydroxypterin; 5-HO-trp, 5-hydroxytryptophan. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 September 7. (16,17). Our present understanding of this mechanism has come primarily from spectroscopy of enzyme-substrate or enzyme-inhibitor complexes and steady-state kinetics. The changes in the ligands to the iron as substr...

Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH4) and O2. A complete kinetic mechanism for PheH was determined by global analysis of single turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH4-phenylalanine complex begins with the rapid binding of BH4 (Kd = 65 µM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (Kd = 130 µM) is approximately ten-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O2 rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, detectable as a decrease in absorbance at 340 nm, with a rate constant of 140 s−1. Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is ten-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines kcat. Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.

The non-heme iron enzyme phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase catalyze the hydroxylation of their aromatic amino acid substrates using a tetrahydropterin as the source of electrons. The hydroxylating intermediate is proposed to be an Fe (IV)O species. We report here that all three enzymes will catalyze hydroxylation reactions using H 2 O 2 in place of tetrahydropterin and oxygen, forming tyrosine and 3-hydroxyphenylalanine from phenylalanine, 4-HOCH 2 -phenylalanine from 4-CH 3 -phenylalanine, and hydroxycyclohexylalanine from 3-cyclohexylalanine. No peroxide-dependent reaction is seen with active site mutants of TyrH and PheH in which the stability or reactivity of the iron center is compromised. These results provide further support for an Fe(IV)O hydroxylating intermediate.Phenylalanine hydroxylase (PheH), tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (TrpH) are non-heme iron monooxygenases that catalyze the insertion of an oxygen atom from O 2 into the aromatic side chain of their corresponding substrates using a tetrahydropterin (PH 4 ) substrate as the reductant. 1-3 Their active sites each contain a mononuclear iron coordinated by two histidines and a glutamate, 4-6 an arrangement that has been termed a 2-his-1-carboxylate facial triad. 7,8 Scheme 1 shows the chemical mechanism proposed for PheH, TyrH, and TrpH. 3 The hydroxylating intermediate is an Fe(IV)O capable of aromatic, benzylic, and aliphatic hydroxylation. 9,10 This species has recently been detected in TyrH by freezequench Mössbauer spectroscopy. 11 The spectra and reactivity of the Fe(IV)O intermediate resemble those in members of the α-ketoglutarate-dependent hydroxylase family, which also contain a mononuclear iron coordinated by a 2-his-1-carboxylate facial triad. 12,13In Scheme 1 the PH 4 supplies two electrons to reduce one atom of O 2 to the level of water, but it plays no role in the actual oxygen transfer to the amino acid substrate. This suggests that it could be possible to bypass the PH 4 and generate the Fe(IV)O intermediate directly with an alternative oxygen donor. Such a shunt has been possible in the cases of the heme-based cytochrome P450, 14 the binuclear non-heme methane monooxygenase, 15 and mononuclear non-heme dioxygenases, 16,17 but not with a mononuclear non-heme monooxygenase.We now report that H 2 O 2 can replace PH 4 and O 2 to support amino acid hydroxylation by the aromatic amino acid hydroxylases. Incubation of PheH,18,19 TyrH,20 or TrpH 19,21 with phenylalanine and H 2 O 2 results in the formation of tyrosine and 3-HO-phenylalanine ( Figure 1). No hydroxylated amino acids are detectable if apoenzyme is used. The rate of hydroxylation is unchanged when the reaction is carried out in the absence of O 2 . The ratio of tyrosine to 3-HO-phenylalanine produced is different for the three enzymes, with ratios of 1.5, 1.2, and 1.6 for PheH, TyrH and TrpH, respectively. Controls showed that the PheH does not lose activity TyrH has previously been shown to produce ty...