Intrinsic deuterium isotope effects on benzylic hydroxylation by tyrosine hydroxylase

Article abstract of DOI:10.1021/ja025602s

Tyrosine hydroxylase (TyrH) is a mononuclear, non-heme iron monooxygenase that catalyzes the pterin-dependent hydroxylation of tyrosine to dihydroxyphenylalanine. When 4-methylphenylalanine is used as a substrate for TyrH, 4-hydroxymethylphenylalanine is

Full text of DOI:10.1021/ja025602s

Published on Web 03/26/2002
Intrinsic Deuterium Isotope Effects on Benzylic Hydroxylation by Tyrosine
Hydroxylase
Patrick A. Frantom,† Rongson Pongdee,‡ Gary A. Sulikowski,‡ and Paul F. Fitzpatrick*,†,‡
Department of Biochemistry and Biophysics, and Department of Chemistry, Texas A&M UniVersity,
College Station, Texas 77843
Received January 15, 2002
Scheme 1
Tyrosine hydroxylase (TyrH) catalyzes the pterin-dependent
hydroxylation of tyrosine to dihydroxyphenylalanine.1-3 This
enzyme is a non-heme, mononuclear ferrous iron monooxygenase
that belongs to the aromatic amino acid hydroxylase family. This
family also includes phenylalanine hydroxylase (PheH) and tryp-
tophan hydroxylase (TrpH). When 4-methylphenylalanine is used
as a substrate for TyrH, three products are produced: 3-hydroxy-
4-methylphenylalanine, 3-methyl-4-hydroxyphenylalanine, and 4-hy-
droxymethylphenylalanine.4 4-Hydroxymethylphenylalanine results
from benzylic hydroxylation as opposed to the aromatic hydroxy-
lation seen with the natural substrate and with the other two
two secondary isotope effects (PS2). With the dideuterated substrate,
products. PheH also catalyzes the benzylic hydroxylation of
removal of a hydrogen exhibits two secondary effects (S2), while
4-methylphenylalanine,5 and TrpH will catalyze the hydroxylation
removal of a deuterium exhibits a primary effect and a secondary
isotope effect (PS). Likewise, the monodeuterated substrate will
show an intrinsic effect equal to a secondary isotope effect when
a hydrogen atom is removed and a primary isotope effect when a
of the methyl group of 5-methyltryptophan.6 While much work has
been done to elucidate the mechanism of aromatic hydroxylation
by these enzymes,1,3,7 the mechanism of benzylic hydroxylation has
not been analyzed.
deuterium is removed. Equations 2 and 3 show the relationships
To examine the mechanism of benzylic hydroxylation by TyrH
between the isotopic composition of the 4-hydroxymethylphenyl-
we have determined the intrinsic primary and secondary deuterium
alanine product and the primary and secondary isotope effects for
isotope effects on this reaction. When 4-methylphenylalanine is used
each substrate. Here, k is the microscopic rate constant for the
abstraction of either a deuterium or a hydrogen atom. The
as a substrate for TyrH, 17.7 ( 0.6% of the amino acid products
result from benzylic hydroxylation.8 The relative amount of benzylic
superscript atoms before the dash describe the remaining atoms,
hydroxylation decreases as the deuterium content of the substrate
and the atom after the dash is the abstracted atom.
methyl group increases (Table 1).9,10 The effects of isotopic
substitution on the relative amounts of the different amino acid
4-CDDOH-phe
4-CDHOH-phe
kDD-H
1 kDD-H kHH-H
P
2S
products can be analyzed by the mechanism of Scheme 1, where
E-X is the hydroxylating species. Here, k1 is the rate constant for
benzylic hydroxylation, and k2 is the sum of the net rate constants
for aromatic hydroxylation at the 3- and 4-positions. The percentage
of 4-hydroxymethylphenylalanine produced is k1/(k1 + k2). Since
k1 is the only step that is sensitive to isotopic substitution on the
methyl group,11 the isotope effect on the percent of benzylic
)
)
)
)
(2)
(3)
(
(
)(
)
)
2kHD-D
kHH-H kHD-D
2
4-CDHOH-phe 2kHD-H
4-CHHOH-phe
kHD-H kHH-H
2P
S
)
) 2
)(
kHH-D
kHH-H kHH-D
Using eqs 2 and 3 and the isotope effect of 14.0 for the
trideuterated amino acid, the intrinsic primary and secondary
deuterium isotope effects were determined from the deuterium
content of the hydroxymethylphenylalanine produced from either
the monodeuterated or the dideuterated amino acid.12 These values
are given in Table 1. The results from the two different substrates
are consistent and give an average intrinsic primary deuterium
isotope effect of 9.6 ( 0.9 and an average intrinsic secondary
deuterium isotope effect of 1.21 ( 0.08.
hydroxylation is related to the intrinsic isotope effect (Dk1 ) k1H
1D) by eq 1. The value of k1/k2 for the nondeuterated substrate is
/
k
0.22 ( 0.01. Thus, the intrinsic deuterium isotope effects on
benzylic hydroxylation can be calculated for each of the deuterated
substrates (Table 1).
k1H/(k1H + k2) Dk1 + k1H/k2
D(% 4-CH2OH-phe) )
)
(1)
1 + k1H/k2
k1D/(k1D + k2)
The large normal secondary deuterium isotope effect is consistent
with significant bond rehybridization at the benzylic carbon during
CH bond cleavage;13 this could arise from a radical or a cationic
species.14 The combination of a large secondary isotope effect and
a primary effect greater than the semiclassical limit has previously
been interpreted as indicating quantum-mechanical tunneling of the
hydrogen.15 Thus, the present results would add TyrH and the other
pterin-dependent hydroxylases to the growing list of enzymes for
which tunneling makes a significant contribution to catalysis.
These intrinsic isotope effects are combinations of primary and
secondary deuterium isotope effects. When the trideuterated
compound is used as a substrate, only a deuterium can be extracted,
so that the intrinsic isotope effect is the product of a primary and
* To whom correspondence should be addressed. E-mail: fitzpat@tamu.edu..
† Department of Biochemistry and Biophysics.
‡ Department of Chemistry.
9
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J. AM. CHEM. SOC. 2002, 124, 4202-4203
10.1021/ja025602s CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Isotope Effects on Benzylic Hydroxylation by Tyrosine Hydroxylase
intrinsic isotope
% benzylic
hydroxylationa
effect on benzylic
hydroxylation
product
isotopic ratiob
intrinsic primary
isotope effect
intrinsic secondary
isotope effect
substrate
4-C2H3-phenylalanine
4-CH2H2-phenylalanine
4-C H2 H-phenylalanine
1.48 ( 0.07
6.6 ( 1.0
12.0 ( 0.8
14.0 ( 1.0
3.0 ( 0.5
1.6 ( 0.2
-
-
-
4.3 ( 0.4
14.5 ( 1.4
10.1 ( 0.7
9.0 ( 0.6
1.18 ( 0.06
1.24 ( 0.06
2
a Percent of amino acid product produced by benzylic hydroxylation. b Ratio of product due to hydrogen abstraction to the product due to deuterium
abstraction as determined by ESI mass spectrometry.
the reaction, 400 µL of 10 mM sodium borate, pH 9.1, was added, followed
by 100 µL 10 mM sodium cyanide and 400 µL 1 mM naphthalene-2,3-
dicarboxaldehyde. The reaction was incubated for 20 min at 30 °C. Fifty
microliters were then injected onto a Waters C18 NovaPak column
equilibrated with 12.5 mM sodium phosphate and 0.5% tetrahydrofuran,
pH 7.0, and eluted with an acetonitrile gradient. Products were detected
by fluorescence with excitation and emission wavelengths of 420 and 490
nm, respectively. Peak areas were quantitated using standard curves from
the authentic products.
Similarly large primary and secondary deuterium effects have
been reported for benzylic hydroxylation by cytochrome P45016
and dopamine â-monooxygenase.17 In the case of dopamine
â-monooxygenase the hydrogen is proposed to be removed as a
hydrogen atom.17,18 The mechanism of cytochrome P450 is
controversial. A mechanism involving hydrogen-atom abstraction
had been widely accepted.19 However, recent analyses of the
probable lifetime of the proposed radical by Newcomb and co-
workers are consistent with multiple iron species as the hydroxy-
lating intermediates producing both radical and cationic interme-
diates.20 Independent of mechanism, in both systems the hydrogen
is thought to be abstracted by a metal-oxo species. A high-valence
iron-oxo species, Fe(IV)dO, has also been proposed as the
hydroxylating intermediate for TyrH.4 The present results support
that proposal and are consistent with the properties of the mono-
nuclear iron site in TyrH resembling those of the heme-based
intermediate in cytochrome P450.
(9) The deuterated amino acids were synthesized using previously described
methods. (a) Reference 4. (b) Lee, Y.; Silverman, R. B. Org. Lett. 2000,
2, 303-306. Each compound was determined to be greater than 97%
deuterated using 1H NMR and mass spectrometry.
(10) When 4-methylphenylalanine is used as a substrate for TyrH, pterin
oxidation is partly uncoupled from amino acid hydroxylation, such that
the amount of amino acid hydroxylation is substoichiometric to the amount
of pterin oxidized.4 No significant change in coupling could be detected
with any of the deuterated substrates.
(11) Isotope effects on binding or on aromatic hydroxylation have not been
explicitly ruled out, but any such effects are expected to be much smaller
than the values reported here.
(12) Negative ion electrospray mass spectrometry was used to determine the
deuterium content of the hydroxymethylphenylalanine. Assays were run
under the same conditions as described in ref 4 except for 300 µM
6-methyltetrahydropterin, and products were separated using a water-
equilibrated column and an acetonitrile gradient. Fractions containing
hydroxymethylphenylalanine were collected and analyzed with negative
ion ESI-TOF mass spectrometry. The ratios of the (m - 1) peaks resulting
from loss or retention of deuterium were used in the calculations.
(13) Klinman, J. P. AdV. Enzymol. Relat. Areas Mol. Biol. 1978, 46, 415-
494.
Acknowledgment. We thank Dr. Shane Tichy from the LBMS
at Texas A&M University for his assistance in acquiring and
interpreting the mass spectrometry data. This work was funded by
NIH Grants R01-GM47291 (P.F.F.) and T32-GM08523 (P.A.F. and
R.P.). The purchase of the ESI mass spectrometer by the LBMS
was supported by the Life Sciences Task Force at Texas A&M.
(14) Removal of the hydrogen as a hydride could generate a significant
secondary isotope effect; however, the primary isotope effect would be
expected to be much smaller than is observed with TyrH. (a) Cha, Y.;
Murray, C. J.; Klinman, J. P. Science 1989, 243, 1325-1330. (b)
Alhambra, C.; Corchado, J. C.; Sanchez, M. L.; Gao, J.; and Trulahr, D.
G. J. Am. Chem. Soc. 2000, 122, 8197-8203.
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