Measurement of intrinsic rate constants in the tyrosine hydroxylase reaction

Article abstract of DOI:10.1021/bi901874e

Tyrosine hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme aromatic amino acid hydroxylase that catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA). Chemical quench analyses of the enzymatic reaction show a burst of DOPA formation, followed by a linear rate equal to the kcat value at both 5 and 30 °C. The effects of increasing solvent viscosity confirm that k_cat is ～84% limited by diffusion, most probably due to slow product release, and that tyrosine has a commitment to catalysis of 0.45. The effect of viscosity on the k_cat/K_m for 6-methyltetrahydropterin is greater than the theoretical limit, consistent with the coupling of pterin binding to the movement of a surface loop. The absorbance changes in the spectrum of the tetrahydropterin during the first turnover, the kinetics of DOPA formation during the first turnover, and the previously described kinetics for formation and decay of the Fe(IV)O intermediate [Eser, B. E., Barr, E. W., Frantom, P. A., Saleh, L., Bollinger, J. M., Jr., Krebs, C., and Fitzpatrick, P. F. (2007) J. Am. Chem. Soc. 129, 11334-11335] were analyzed globally, yielding a single set of rate constants for the TyrH reaction. Reversible binding of oxygen is followed by formation of Fe(IV)O and 4a-hydroxypterin with a rate constant of 13 s^-1 at 5 °C. Transfer of oxygen from Fe(IV)O to tyrosine to form DOPA follows with a rate constant of 22 s^-1. Release of DOPA and/or the 4a-hydroxypterin with a rate constant of 0.86 s^-1 completes the turnover.

Full text of DOI:10.1021/bi901874e

Biochemistry 2010, 49, 645–652 645
DOI: 10.1021/bi901874e
Measurement of Intrinsic Rate Constants in the Tyrosine Hydroxylase Reaction†
Bekir E. Eser‡ and Paul F. Fitzpatrick*,§
‡Department of Chemistry, Texas A&M University, College Station, Texas 77843 and §Department of Biochemistry and Center for
Biomedical Neuroscience, University of Texas Health Science Center, San Antonio, Texas 78229
Received November 2, 2009; Revised Manuscript Received December 17, 2009
ABSTRACT: Tyrosine hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme aromatic amino acid
hydroxylase that catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA). Chemical quench
analyses of the enzymatic reaction show a burst of DOPA formation, followed by a linear rate equal to the kcat
value at both 5 and 30 °C. The effects of increasing solvent viscosity confirm that kcat is ∼84% limited by
diffusion, most probably due to slow product release, and that tyrosine has a commitment to catalysis of 0.45.
The effect of viscosity on the kcat/Km for 6-methyltetrahydropterin is greater than the theoretical limit,
consistent with the coupling of pterin binding to the movement of a surface loop. The absorbance changes in
the spectrum of the tetrahydropterin during the first turnover, the kinetics of DOPA formation during the
first turnover, and the previously described kinetics for formation and decay of the Fe(IV)O intermediate
[Eser, B. E., Barr, E. W., Frantom, P. A., Saleh, L., Bollinger, J. M., Jr., Krebs, C., and Fitzpatrick, P. F.
(2007) J. Am. Chem. Soc. 129, 11334-11335] were analyzed globally, yielding a single set of rate constants for
the TyrH reaction. Reversible binding of oxygen is followed by formation of Fe(IV)O and 4a-hydroxypterin
with a rate constant of 13 s-1 at 5 °C. Transfer of oxygen from Fe(IV)O to tyrosine to form DOPA follows
with a rate constant of 22 s-1. Release of DOPA and/or the 4a-hydroxypterin with a rate constant of 0.86 s-1
completes the turnover.
Tyrosine hydroxylase (TyrH)1 is a member of the pterin-
dependent aromatic amino acid hydroxylase family (1, 2); these
enzymes utilize a mononuclear non-heme iron and a reduced
pterin to activate molecular oxygen for the hydroxylation of their
corresponding amino acid substrates. TyrH, which is found in the
The cationic amino acid intermediate in Scheme 2 was originally
proposed on the basis of an analysis of the kinetics of TyrH with a
series of para-substituted phenylalanines (8). Subsequent mea-
surements of the secondary deuterium kinetic isotope effect on
hydroxylation for all the aromatic amino acid hydroxylases
provided further evidence of an electrophilic aromatic substitu-
tion reaction mechanism (7, 9, 11, 12). Initial indirect evidence for
the Fe(IV)O intermediate came from the observation that the 4a-
HO-6MPH3 product could be formed in the absence of amino
acid hydroxylation, establishing that heterolytic cleavage of the
oxygen-oxygen bond is required to form the hydroxylating
intermediate (7, 13, 14). This set the formal oxidation level of
the hydroxylating intermediate as Fe(IV)O in the absence of
additional electron transfer steps. More recently, rapid freeze
brain and adrenal gland, catalyzes conversion of
L-tyrosine to
L-dihydroxyphenylalanine (DOPA) (Scheme 1). This is the first
and rate-limiting step in the biosynthesis of the catecholamine
neurotransmitters dopamine, noradrenaline, and adrenaline. A
deficiency of TyrH is associated with several neurological dis-
orders, including DOPA-responsive Parkinson’s disease, pro-
gressive encephalopathy, DOPA-nonresponsive dystonia, and
DOPA-responsive dystonia (Segawa’s disease) (3, 4). The other
members of the family are also physiologically significant;
phenylalanine hydroxylase, a liver enzyme, catalyzes the conver-
sion of phenylalanine in the diet to tyrosine, and tryptophan
hydroxylase, a brain enzyme, converts tryptophan to 5-hydro-
xytryptophan as a precursor to the neurotransmitter serotonin.
The members of the aromatic amino acid hydroxylase family
have similar active sites in which the iron atom is coordinated by
the common 2-His-1-Glu facial triad motif (5, 6). Studies of the
three enzymes have established that they share a common
catalytic mechanism (7-12), shown for TyrH in Scheme 2 (1).
€
quench Mossbauer spectroscopy provided direct evidence of an
Fe(IV) species as the hydroxylating intermediate in the TyrH
reaction (15). The initial iron-peroxo-pterin intermediate in
Scheme 2 has not been detected, although E332A TyrH produces
a pterin species with a UV absorbance spectrum consistent with a
4a-hydroperoxypterin (16). Together these results have provided
strong evidence of the intermediates in the mechanism of
Scheme 2. Still, a complete understanding of the reaction of
TyrH requires knowledge of the rate constants for their inter-
conversion. The experiments described here were designed to
determine such rate constants.
†This work was supported by in parts by grants from the National
Institutes of Health (R01 GM047291) and The Welch Foundation (AQ-
1245).
EXPERIMENTAL PROCEDURES
*To whom correspondence should be addressed: Department of
Biochemistry, University of Texas Health Science Center, San Antonio,
TX 78229. E-mail: fitzpatrick@biochem.uthscsa.edu. Phone: (210) 567-
8264. Fax: (210) 567-8778.
Chemicals. 6-Methyltetrahydropterin was from Schircks
Laboratories (Jona, Switzerland). Ferrous ammonium sulfate,
Hepes, sucrose, and trehalose were purchased from Fisher
(Pittsburgh, PA). Glycerol and tyrosine were from Sigma-Aldrich
(Milwaukee, WI). The GEMINI reverse-phase C18 HPLC
1Abbreviations: TyrH, tyrosine hydroxylase; DOPA, dihydroxyphe-
nylalanine; 6MPH4, 6-methyltetrahydropterin; 4a-HO-6MPH3, 4a-
hydroxypterin; DOPA, 3,4-dihydroxyphenylalanine.
r
2009 American Chemical Society
Published on Web 12/21/2009
pubs.acs.org/Biochemistry

646 Biochemistry, Vol. 49, No. 3, 2010
Eser and Fitzpatrick
column was obtained from Phenomenex (Torrance, CA).
All other reagents were of the highest purity commercially
available.
substrate (5 μM to 1 mM). The buffer consisted of 200 mM Hepes
(pH 7.5) and 0.1 M KCl, with or without viscogen. The absolute
viscosities (η) for buffers at 5 and 30 °C were calculated using
values reported in the literature (21-23). The small effects of
buffer and salt on the viscosity of the solutions were neglected.
Chemical Quench. Chemical quench experiments were per-
formed using an SFM-400/Q rapid-mixing instrument from Bio-
Logic (Claix, France) in quenched-flow mode. The instrument
was made anaerobic through incubation with a sodium dithionite
solution (∼100 mM) for at least 2 h. The water bath was bubbled
with nitrogen gas to prevent diffusion of O2 into the system. Apo-
TyrH (30-40 μM) in 200 mM Hepes (pH 7.5), 10% glycerol, and
0.1 M KCl (total volume of ∼10 mL) was made anaerobic in a
tonometer through at least 20 argon-vacuum cycles. Ferrous
ammonium sulfate (∼20 μL, ∼0.9 equiv of enzyme) was then
added to the tonometer under argon. The 6MPH4 stock solution
(∼40 mM) was prepared in 2 mM HCl; an extinction coefficient
of 17.8 mM-1 cm-1 in 2 M perchloric acid was used to determine
the exact concentration. A volume corresponding to a final
concentration in the tonometer of 2 mM 6MPH4 was placed in
the side arm under argon. Additional argon-vacuum cycles were
performed prior to the 6MPH4 solution being mixed with the
tonometer contents, which were then loaded into one of the
syringes of the rapid-mixing instrument. A second syringe was
loaded with buffer containing 500 μM tyrosine that had been
bubbled with pure oxygen gas for at least 20 min. This was done
either on ice to produce an oxygen concentration of 1.9 mM or at
room temperature to produce a concentration of 1.2 mM. The
quenching solution, 5 M HCl, was loaded into a third syringe.
The tonometer contents were mixed with the tyrosine-containing
oxygenated buffer and quenched with acid after being aged
through a 90 μL (N° 3) delay line. Tyrosine and DOPA were
separated on a Phenomenex C18 HPLC column (250 mm ꢀ
4.6 mm) with an isocratic mobile phase of 15 mM sodium
phosphate (pH 7.0) at a flow rate of 1 mL/min. The amount of
DOPA was quantified using a Waters 2475 Multi λ fluorescence
detector with an excitation wavelength of 270 nm and an emission
wavelength of 310 nm.
Expression and Purification of TyrH. Rat TyrH was
expressed in Escherichia coli and purified as previously de-
scribed (17, 18), with the exceptions that cell cultures were grown
in LB broth containing 500 μM FeCl3 and 2 mM MgSO4 and
growth continued for ∼20 h at 20 °C after induction with isopropyl
β-thiogalactoside. To remove the ferric iron present in the enzyme
as purified, enzyme to be used in the rapid reaction analyses was
dissolved in 200 mM Hepes (pH 7.5), 10% glycerol, and 0.1 M
KCl containing 5 mM EDTA and incubated for 1 h on ice before
being dialyzed against the same buffer without EDTA. The
iron content was determined using a Perkin-Elmer Analyst 700
atomic absorption spectrophotometer (19). For experiments in
which viscogen was used, the enzyme was dialyzed into buffer
that contained no glycerol [200 mM Hepes (pH 7.5) and 0.1 M
KCl].
Steady-State Kinetics. Steady-state kinetic parameters of
TyrH were determined using a colorimetric end point assay to
measure DOPA production, as previously described (20). Con-
ditions were 0.1-0.5 μM TyrH, 100 μg/mL catalase, 1 mM
dithiothreitol (DTT), 10 μM ferrous ammonium sulfate, and
either 1 mM 6MPH4 when tyrosine was the varied substrate
(3-300 μM) or 200 μM tyrosine when 6MPH4 was the varied
Scheme 1
Scheme 2

Article
Stopped-Flow Spectrophotometry. Single-turnover ki-
Biochemistry, Vol. 49, No. 3, 2010 647
netics of TyrH were monitored using an Applied Photophysics
(Leatherhead, U.K.) SX20 stopped-flow spectrophotometer in
absorbance mode. The instrument was made anaerobic in the
same way as described above for the chemical quench experi-
ment. A solution of apo-TyrH (200 μM) and tyrosine (550 μM) in
200 mM Hepes (pH 7.5) and 0.1 M KCl was made anaerobic in a
tonometer through at least 20 argon-vacuum cycles. A stoichio-
metric amount of ferrous ammonium sulfate (∼20 μL, ∼0.9 equiv
of enzyme) was then added to the tonometer under argon. A
6MPH4 solution, corresponding to a final concentration of 1.1 mM,
was placed in the side arm of the tonometer under argon.
Additional argon-vacuum cycles were performed before the
6MPH4 solution was mixed with the tonometer contents. The
tonometer was then loaded into one of the syringes of the
stopped-flow instrument. The second syringe was loaded with
the same buffer containing 120 μM oxygen at 5 °C.
Data Analysis. The kinetics of DOPA formation from the
chemical quench experiments were analyzed using the global
analysis program KinTek Explorer Pro, which utilizes direct
numerical integration to simulate experimental results (KinTek
Corp., Austin, TX) (24). SpecFit (25, 26) (Spectrum Software
Associates) was used for initial analysis of stopped-flow data at
multiple wavelengths by singular-value decomposition. Subse-
quent global analyses were conducted with KinTek Explorer.
Error analysis was done with the FitSpace Explorer option of
KinTek Explorer (27) and a threshold value of 1.2 sum square
error (SSE). Igor Pro (Wavemetrics, Lake Oswego, OR) was used
for analysis of the effects of viscosity on the steady-state kinetics
using eqs 1-3.
F
IGURE 1: Time course at (A) 5 or (B) 30 °C for the formation of
DOPA from the reaction of TyrH (30-40 μM) and 6MPH4 (2 mM)
with an equal volume of Tyr (500 μM) and O2 (1.9 mM at 5 °C and
1.2 mM at 30 °C). The lines are from fits to the mechanism in
Scheme 3 with values for kburst (pseudo-first-order rate constant with
respect to oxygen concentration) and klinear of 7.0 ( 2.4 and 0.65 (
0.05 s-1, respectively, for the data at 5 °C and of 18.8 ( 5.4 and 3.5 (
0.5 s-1, respectively, for the data at 30 °C. The line in the inset of panel A
is based on the mechanism in Scheme 4, with the rate constants listed
in Table 2.
RESULTS
Consequently, the extent to which product release limits turnover
will be reflected in the sensitivity of the kcat value to the viscosity
of the medium. Entry of the substrate into the active site is
similarly expected to be diffusion-limited, so that the effects
of viscosity on kcat/Km values will reflect the magnitude of the
rate constant for the initial binding relative to those for subsequent
steps up to and including the first irreversible step (30-33, 35).
Glycerol, sucrose, and trehalose were used as viscogens to
determine the effect of solvent viscosity on the steady-state kinetic
parameters for TyrH. However, in initial experiments, the effects
of glycerol were nonlinear and suggested that glycerol directly
inhibited the enzyme at high concentrations. In fact, PheH has
been reported to be inhibited by glycerol (36). Therefore, more
complete analyses were conducted with only sucrose and treha-
lose as viscogens.
The effects of sucrose and trehalose on the steady-state kinetic
parameters for TyrH are illustrated in Figures 2-4. The data are
plotted as the ratio of the relevant kinetic parameter in the ab-
sence of viscogen to that in its presence as a function of the
relative viscosity of the solvent. The effect of viscosity is the slope
of such a plot and can take values between 0 and 1. A viscosity
effect of 0 means the rate of the reaction is completely indepen-
dent of solvent viscosity, whereas an effect of 1 indicates a
completely diffusion-limited event. To analyze the data, the
initial rates as a function of substrate concentration at each
concentration of viscogen were fit to eq 1 to yield the viscosity
effects on both kcat and kcat/Km simultaneously
Chemical Quench Experiments. The kinetics of DOPA
formation during the first few turnovers of the TyrH reaction
were analyzed by chemical quench methods. To do so, the
anaerobic complex of ferrous TyrH (30-40 μM) with 6MPH4
(2.0 mM) was mixed with an equal volume of buffer containing
tyrosine (500 μM) and O2 (1.9 mM at 5 °C and 1.2 mM at 30 °C).
The tyrosine, oxygen, and 6MPH4 concentrations were set
sufficiently high to achieve complete and rapid binding
(> 15 Km). The reaction was quenched with 5 M HCl at times
from 10 ms to several seconds, and the samples were analyzed by
HPLC to quantify the amount of the product DOPA. The time
courses at 5 and 30 °C are shown in Figure 1. An initial burst can
be clearly seen at both temperatures. For quantitative analysis,
the data were fit to the mechanism in Scheme 3 using KinTek
Explorer; here kburst represents the rate constant for the forma-
tion of DOPA during the first turnover, and klinear represents the
rate of DOPA formation during subsequent turnovers and should
match kcat. Binding of substrates was assumed to be rapid and was
not included in the analysis. The data at 5 °C could be fit with rate
constants of 7.0 ( 2.4 s-1 for the burst phase and 0.65 ( 0.05 s-1
for the linear phase. The fit of the data at 30 °C yields values for
k
burst and klinear of 18.8 ( 5.4 and 3.5 ( 0.5 s-1, respectively.
Steady-State Viscosity Experiments. The chemical quench
analyses establish that the chemical steps in the TyrH reaction
that result in DOPA formation are significantly faster than
the following steps. These slower steps are frequently product
release or conformational changes associated with product
release (28-35). Product release should be diffusion-limited, with
a rate constant dependent on the viscosity of the medium (33).
kcat
S
v ¼
ð1Þ
Km½1 þ mðμÞꢁ þ S½1 þ nðμÞꢁ

648 Biochemistry, Vol. 49, No. 3, 2010
Eser and Fitzpatrick
Scheme 3
F
IGURE 3: Effectof solvent viscosity onthe kcat/Km value for tyrosine
at (A) 5 and (B) 30 °C for sucrose (b) or trehalose (O) as the viscogen.
The solid linesare based onthe averagesofthe viscosityeffects ateach
temperature for sucrose and trehalose determined from eq 2 (A) or eq
1 (B). The dashed lines show the theoretical limits for a completely
diffusion limited reaction.
F
IGURE 2: Effect of solvent viscosity on the kcat value at (A) 5 and
(B) 30 °C for sucrose (b) or trehalose (O) as the viscogen. The solid lines
are based on the averages of the viscosity effects at each temperature for
sucrose and trehalose determined from eq 1. The dashed lines show the
theoretical limits for a completely diffusion limited reaction.
kcat/K6MPH is much greater at 30 °C than at 5 °C. Only at 5 °C is
where μ is the relative viscosity (η/η0)2 minus 1 and m and n are
the viscosity effects on kcat/Km and kcat, respectively. Equation 2
was used instead of eq 1 to calculate the viscosity effects on the
kcat/Km value for tyrosine at 5 °C, using only the initial rate data
obtained at low tyrosine concentrations.
4
the viscosity effect within the expected range of 0-1. In all the
other cases, the viscosity effect is greater than 1 (Figure 4B and
Table 1). Such deviations from the expected limit have previously
been attributed to internal motions in proteins occurring con-
comitantly with binding (28, 37-39).
kcat
S
Single-Turnover Stopped-Flow Experiments. As a com-
plementary approach to determining rate constants for indivi-
dual steps in the TyrH reaction, stopped-flow absorbance
spectroscopy was used to monitor the pterin intermediates that
form during the reaction of TyrH. The non-heme iron center of
TyrH is colorless, although there is a weak charge transfer
absorbance band in the near UV (19), ruling out the possibility
of monitoring the reaction by following changes in the absor-
bance spectrum of the iron. However, several oxidized and
reduced forms of pterin have distinct absorbance spectra
at 200-450 nm (7). An anaerobic TyrH (200 μM)/6MPH4
(1.1 mM)/Tyr (550 μM) solution was mixed with an equal volume
of oxygenated buffer (120 μM) at 5 °C. Under these conditions,
the concentration of oxygen limits the reaction to a single
turnover. Figure 5 shows the resulting absorbance changes at
246 and 318 nm. The 4a-hydroxypterin product absorbs maxi-
mally at 246 nm, while the absorbance changes at 318 nm reflect
both the formation of the hydroxypterin product and its sub-
sequent dehydration to quinonoid dihydropterin (7). The absor-
bance traces at both wavelengths were fit simultaneously to
v ¼
ð2Þ
Km½1 þ mðμÞꢁ
The results are summarized in Table 1.
Increasing the viscosity of the solution with either sucrose or
trehalose results in a significant decrease in the kcat value at both 5
and 30 °C (Table 1 and Figure 2). The effect at both temperatures
is independent of the identity of the viscogen, with an average
effect of 0.84 at 5 °C and 0.88 at 30 °C. This indicates that the kcat
value is significantly limited by a diffusional event. Sucrose and
trehalose also have similar effects on the kcat/Km value for
tyrosine at both 5 and 30 °C, with average values of 0.44 and
0.42 at the two temperatures (Figure 3). The values suggest that
the kcat/Km value for tyrosine is ∼45% limited by diffusion of
tyrosine into the active site. The effects of increased viscosity on
the kcat/Km value for 6MPH4 are more complex. The effect varies
with both the temperature and identity of the viscogen (Figure 4
and Table 1). For both sucrose and trehalose, the effect on
2Parameters denoted with a superscript zero, such as kcat0 or η0, refer
to the values in the absence of viscogen.

Article
Biochemistry, Vol. 49, No. 3, 2010 649
Table 1: Effects of Viscosity on the Steady-State Kinetic Parameters for
TyrHa
b
kcat
c
b
viscogen
temp (°C)
kcat/Ktyr
kcat/K6MPH
4
sucrose
5
5
0.89 ( 0.09
0.78 ( 0.07
0.83 ( 0.03
0.94 ( 0.10
0.49 ( 0.09
0.38 ( 0.10
0.30 ( 0.11
0.54 ( 0.15
1.41 ( 0.26
0.27 ( 0.10
1.93 ( 0.27
1.26 ( 0.29
trehalose
sucrose
30
30
trehalose
aConditions: 0.1-0.5 μM TyrH, 100 μg/mL catalase, 1 mM DTT, 10 μM
ferrous ammonium sulfate, 200 mM Hepes (pH 7.5), and 0.1 M KCl. bWith
200 μM tyrosine with varied concentrations of 6MPH4 (5 μM to 1 mM).
cWith 1 mM 6MPH4 with varied concentrations of tyrosine (3-300 μM).
F
IGURE 5: Stopped-flow absorbance traces at 246 (O) and 318 nm
(0) for the reaction of a 100 μM TyrH/Fe(II)/6MPH4/Tyr solution
with 60 μM O2. The solid lines werecalculated from the mechanism in
Scheme 4 using the rate constants and extinction coefficients listed in
Table 2.
F
IGURE 4: Effect of solvent viscosity on the kcat/Km value for 6MPH4
at (A) 5 and (B) 30 °C for sucrose (b) or trehalose (O) as the viscogen.
The solid lines are linear regression fits with slopes corresponding to
the viscosity effects reported in Table 1. The dashed lines show the
theoretical limits for a completely diffusion limited reaction.
global analysis. Estimates for the extinction coefficients of free
6MPH4 and 4a-HO6MPH3 were also taken from previous
studies (7, 16). To obtain estimates of the extinction changes
for enzyme-bound pterin species and of the concentrations of
active enzyme, these values were adjusted manually by taking
advantage of the live simulation display option of KinTek
Explorer (24). The resulting fraction of active enzyme was at
various sequential mechanisms using SpecFit (25, 26). A three-
step sequential mechanism, with rate constants of 15, 0.7, and
0.024 s-1 accounted well for the data at both wavelengths. This
analysis provided estimates of the rate constants for formation of
the bound hydroxypterin from the reaction of oxygen with the
enzyme complex, for a subsequent step that resulted in a further
spectral change, and for the slow dehydration of the hydroxyp-
terin to the quinonoid dihydropterin.
Global Analyses. The stopped-flow results in Figure 5 pro-
vide estimates of the rate constants for steps in which the
absorbance of the pterin substrate changes significantly. The
chemical quench experiments in Figure 1 provide rate constants
for hydroxylation of the amino acid substrate and for kcat. The
€
least 95%, except for the freeze quench Mossbauer data, where
the fraction of active enzyme concentration was 80% (16).
Finally, a global fit to all three data sets was conducted to yield
values of k2, k3, and k4. The confidence intervals for the resulting
values were determined using the FitSpace Explorer option of
KinTek Explorer (27). FitSpace determines estimates of the
confidence intervals of fitted parameters by determining the
dependence of the sum square error (SSE) on each pair of
parameters while allowing all other parameters to be varied (27).
The best-fit values for the fitted rate constants, their confidence
intervals (lower and upper boundaries), and extinction coeffi-
cients from the global fitting analysis are listed in Table 2. The
values of k2, k4, and k5 agree well with the results of analyzing the
€
earlier freeze quench Mossbauer data (15) provide rate constants
for formation and decay of the Fe(IV) intermediate. To integrate
the results of the three analyses into a single kinetic mechanism,
global analysis was conducted using all the single-turnover data
at 5 °C. The data from all the experiments were fit simultaneously
to the mechanism in Scheme 4 using KinTek Explorer Pro
(KinTek Corp.) (24). Scheme 4 describes a minimal mechanism
of four steps plus a fifth step to account for the hydrolysis of the
4a-HO-6MPH3 product. It was necessary to include an initial
step for oxygen binding because the individual experiments were
conducted at different concentrations of oxygen. We have
previously analyzed the oxygen dependence of the absorbance
changes that occur upon oxygen binding (16); the rate constants
for the reversible oxygen binding step (k1 and k-1) from that
study were used for our global analysis. The rate constant of
0.024 s-1 obtained from the final decrease in absorbance at 246 nm
and increase at 318 nm in the stopped-flow experiment is
consistent with reported values for nonenzymatic dehydration
of a 4a-hydroxypterin to give quinonoid dihydropterin in solu-
tion (7, 13, 40, 41), so that this rate constant was not varied in the
stopped-flow data alone. The value of k4 agrees well with kcat
,
consistent with slow product release, while the value of kburst is
within error of the net rate constant for the first three steps,
consistent with hydroxylation occurring with rate constant k3.
€
Analysis of the Mossbauer data alone previously gave rate
constants for formation and decay of the Fe(IV) species compar-
able to the values of k2 and k3. To illustrate the agreement
between the data and the kinetic model, the data in Table 2 were
used to generate the lines in Figures 5 and 6 and in the inset of
Figure 1A.
DISCUSSION
Because TyrH and the other aromatic amino acid hydroxy-
lases lack a chromophoric cofactor, measurement of individual
rate constants poses a significant challenge. However, individual

650 Biochemistry, Vol. 49, No. 3, 2010
Eser and Fitzpatrick
Scheme 4
Table 2: Values of the Rate Constants, Their Confidence Intervals, and Associated Extinction Changes Obtained from Global Fitting of the Data in
€
Figures 1A and 5 and the Mossbauer Time Course from ref 15, All at 5 °C
rate constant
best-fit value
lower bound
upper bound
Δε246 (mM-1 cm-1
)
Δε318 (mM-1 cm-1
)
k1
300 mM-1 s-1a
50 s-1a
-
-
-
-
-
-
-
-
-0.4
-
-0.7
1.5
k-1
k2
12.7 s-1
9.3 s-1
14.4 s-1
0.54 s-1
-
17.7 s-1
44.0 s-1
1.55 s-1
-
2.0
k3
22.5 s-1
-
8.0
-6.0
k4
0.86 s-1
k5
0.024 s-1b
aTaken from ref 16 and kept fixed. bDetermined from the stopped-flow data alone and kept fixed.
€
rapid quench Mossbauer data in the first 30 ms do not themselves
€
allow reliable estimation of the value of k2. While Mossbauer
spectroscopy is the most direct measure of the Fe(IV)O inter-
mediate, it is also the least precise. Such analyses are expensive in
spectrometer time and enzyme, limiting the number of data
points, and it is not possible to freeze the reaction rapidly enough
on the millisecond time scale to obtain high accuracy at these
early time points. Still, these data show directly at what times the
Fe(IV) intermediate is present, placing important constraints on
the rate constants for its formation and decay. The next step is
hydroxylation of the amino acid by the Fe(IV)O with rate
constant k3. This step does not correspond to a change in the
absorbance spectrum of the pterin bound to the enzyme. How-
ever, the chemical quench analysis of DOPA formation estab-
lishes that DOPA is formed in this step, and the freeze quench
F
IGURE 6: Comparison of predicted concentrations of the Fe(IV)O
intermediate with the experimentally determined concentrations. The
line was calculated from the mechanism of Scheme 4 with the rate
constants in Table 2. The points are taken from ref 15.
€
Mossbauer experiment establishes that Fe(IV)O decays in this
rapid quench analyses of the Fe(IV) intermediate and the amino
acid product and stopped-flow spectroscopic analyses of changes
in the spectrum of the pterin substrate each allow measurement of
a subset of the rate constants in Scheme 4, and the combination of
results from the different approaches means the values for the
rate constants of interest, k2, k3, and k4, are all based on more
than one experimental approach. The global analysis described
here of the results from separate, chemical quench, freeze quench,
and stopped-flow experiments yields values of these rate con-
stants consistent with all the data. The first step in Scheme 4 is
reversible binding of oxygen, with no detectable absorbance
change upon oxygen binding. The rate constants for this step
were determined previously by analyzing the effect of the oxygen
concentration on the absorbance changes in stopped-flow ana-
lyses (16). The second step corresponds to an increase in
absorbance at 246 nm and a decrease at 318 nm as the 4a-
hydroxypterin forms. In the mechanism of Scheme 2, formation
of the 4a-hydroxypterin accompanies formation of Fe(IV)O.
step. The fourth step is assigned to product release on the basis of
the agreement of k4 with kcat and the conclusion from both the
viscosity effects and the chemical quench analysis that product
release limits turnover. The final step with rate constant k5 is
required to account for the absorbance changes seen in the
stopped-flow experiment at longer times. This is a nonenzymatic
step; the rate constant for k5 in Table 2 agrees well with previous
analyses.
The absorbance changes for the second step in Scheme 4 are
less than expected for the formation of 4a-hydroxypterin at
neutral pH (7). However, the sum of the absorbance changes
for the second and fourth steps at both 246 and 318 nm is
consistent with the overall formation of 4a-hydroxypterin from
6MPH4. This suggests that the spectrum of 4a-hydroxypterin
within the enzyme active site is perturbed. Only upon release of
the pterin from the enzyme is the typical absorbance spectrum of
a 4a-hydroxypterin seen. The agreement of k4 with the rate
constant for the linear rate of the chemical quench analysis and
the kcat value establishes this as the rate constant for product
€
This is confirmed by the Mossbauer time course, although the

Article
Biochemistry, Vol. 49, No. 3, 2010 651
6MPH4. The dependence of the effect of viscosity on the
kcat/Km for 6MPH4 on the viscogen and temperature implies
that there are differences in the interaction of sucrose
and trehalose molecules with the flexible loop. Sucrose and
trehalose have previously been reported to interact differen-
tly with water and proteins (47-49). Sucrose has been repor-
ted to make more hydrogen bonding interactions with proteins
(48-50).
Table 3: Values of Association Rate Constants and Forward Commit-
ments for Tyrosine Binding Calculated from the Effects of Viscosity on
k
cat/Ktyr
temp (°C)
kon (μM-1 s-1
)
kf/koff
5
1.22 ( 0.32
7.0 ( 2.0
1.03 ( 0.62
0.93 ( 0.60
30
Overall, this study provides further insight into the mechanism
of TyrH. Combining results from several kinetic analyses has
allowed measurement of rate constants for individuals steps in
the reaction. Turnover is limited by product release. Tyrosine has
a moderate commitment to catalysis, while binding of the
tetrahydropterin is coupled to protein motion.
release. This suggests that the physical step that mainly deter-
mines the kcat value for TyrH is the release of the 4a-hydro-
xypterin, although the data presented here do not rule out a
contribution of DOPA release to rate limitation. Another
possibility is a rate-limiting conformational change associated
with the release of 4a-hydroxypterin, since the viscosity data
showed the presence of a rate-limiting conformational change in
the case of 6MPH4 binding (see below).
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expected viscosity effects on kcat (31, 35), yielding values of
0.95 at 5 °C and 0.85 at 30 °C. These are very similar to the
experimental values obtained from the analysis of the steady-
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