Wild type rabbit tryptophan hydroxylase
(TRH) and two truncated mutant proteins have been expressed in
Escherichia coli. The wild type protein was only expressed
at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion
bodies. The protein that also lacked the carboxyl-terminal 28 amino
acids, TRH102-416, was expressed as 30% of total cell
protein. Analytical ultracentrifugation showed that
TRH102-416 was predominantly a monomer in solution. The
enzyme exhibited an absolute requirement for iron (ferrous or ferric)
for activity and did not turn over in the presence of cobalt or copper.
With either phenylalanine or tryptophan as substrate, stoichiometric
formation of the 4a-hydroxypterin was found. Steady state
kinetic parameters were determined with both of these amino acids using
both tetrahydrobiopterin and 6-methyltetrahydropterin.
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INTRODUCTION |
Tryptophan hydroxylase
(TRH1, EC 1.14.16.4) carries
out the 5-hydroxylation of tryptophan via the oxidation of
tetrahydropterin and the reductive incorporation of molecular oxygen
(Scheme 1). In mammalian metabolism the reaction catalyzed by TRH
precedes
-decarboxylation and is believed to be the initial and
rate-limiting process in the production of the neurotransmitter
serotonin (5-hydroxytryptamine). Although TRH has been studied since
the early 70s, enzymological characterization has been impeded by the
limited quantity of active enzyme available from native or heterologous
sources, the exceedingly low specific activity of the isolated enzyme,
and the quite rapid decrease in activity observed during purification
or storage (1-9).
TRH is a member of the small family of pterin-dependent
aromatic amino acid hydroxylases that includes tyrosine hydroxylase (TYH) and phenylalanine hydroxylase (PAH). Each of these enzymes catalyzes the addition of an oxygen atom to the ring of an aromatic amino acid substrate. The bulk of what is currently known of the reaction mechanism of these enzymes has come from studies of the latter
two (10). Both PAH and TYH require ferrous iron for activity (11, 12);
however, the exact role for the iron in catalysis is undefined. The
primary structures of these enzymes are known from a variety of
organisms. Sequence comparisons and deletion mutageneses have
identified three functional regions: an amino-terminal regulatory
domain, a catalytic domain, and a carboxyl-terminal interface (13-17).
The regulatory domains of the three hydroxylases show no similarities,
whereas the catalytic domains are homologous, with sequence identities
of 32-75%. Enzymes lacking the regulatory domain are catalytically
active (13, 14, 17). The carboxyl-terminal 24 amino acids of TYH form a
long helix demonstrated to be responsible for the tetrameric structure
of the enzyme (13, 18); this helix is presumed to have a corresponding
function in TRH and PAH.
We report here the purification and preliminary characterization of a
mutant protein containing only the catalytic core of TRH. The rationale
for the truncations was to increase the heterologous expression and/or
stability of the enzyme by removing both the regulatory and interface
domains. This doubly truncated form of the enzyme serves as the first
viable model enzyme for detailed mechanistic studies of the catalytic
reaction mechanism of TRH. The high specific activity of this enzyme
has allowed the analysis of several fundamental properties of this
important enzyme, including catalytic specificity and metal
dependence.
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EXPERIMENTAL PROCEDURES |
Materials--
Tryptophan, 5-hydroxytryptophan, and
-mercaptoethanol were obtained from Sigma. 6-Methyltetrahydropterin
was synthesized according to Fitzpatrick (19). Tetrahydrobiopterin was
purchased from Calbiochem. MES and dithiothreitol were purchased from
Research Organics, Inc. Catalase was purchased from Boehringer
Mannheim. Ceramic hydroxyapatite was from Bio-Rad, and Q-Sepharose was
obtained from Amersham Pharmacia Biotech. Isopropyl-
-thiogalactoside
was from United States Biochemical Corp. Cuprous chloride, cupric sulfate, ferric sulfate, and ferrous ammonium sulfate were from Sigma.
Cobalt chloride was purchased from Mallinkrodt. Agarose (SeaKem) was
from FMC. [3,5-3H]Tyrosine was from Amersham Pharmacia
Biotech. Ultma polymerase and deoxynucleotides for polymerase chain
reaction were obtained from Perkin-Elmer. Plasmid pET3d and
Escherichia coli BL21 (DE) were obtained from
Novagen. E. coli CJ236 was from Invitrogen. Plasmid pTZ-18R
was from Amersham Pharmacia Biotech. Oligonucleotides were
custom-synthesized using an Applied Biosystems model 380B synthesizer.
Restriction and DNA modification enzymes were purchased from New
England Biolabs. Plasmids were purified using the Qiagen midi-prep
plasmid preparation kit.
Vectors for TRH Expression--
The construct for expression of
wild type TRH was made by polymerase chain reaction subcloning from the
rabbit cDNA-derived plasmid prbTRH479 (20). NcoI and
BamHI restriction sites were incorporated at the 3' and 5'
ends of the gene, respectively, via non-complementary oligonucleotide
tails. The 1.33-kilobase pair product was then digested and subcloned
into pET3d and pTZ18R to form the wild type TRH constructs pEWOH2 and
pWH1 for expression and mutagenesis, respectively. In pEWOH2 the start
codon for the TRH gene is 6 bases from its ribosome binding site and 55 bases from the T7 promoter.
Deletion of the amino-terminal 101 amino acids of TRH was achieved
using the Bio-Rad adaptation of the methods of Kunkel et al.
(21) with single-stranded uracil-containing DNA derived from pWH1. The
oligonucleotide 5'
ATGAAGGAAGAAGCCATGGAGAGTGTTCCTTGGTTTCCA 3' was used to
incorporate an NcoI restriction site (in bold) into the TRH
gene adjacent to position 101. The resulting plasmid was digested with
NcoI and BamHI and ligated into pET3d to obtain pEWOH
101. The start codon/ribosome binding site/promoter
relationship of pEWOH
101 was unchanged from that in pEWOH2.
Exclusion of the carboxyl-terminal interface helix from translation was
achieved using a variation of the Stratagene quick change mutagenesis
method. pEWOH
101 was used as a template for a polymerase chain
reaction reaction in which two complementary oligonucleotides (5'
GCCAAAAGCTAAACGAATGCCTAAAACGAGCTGC 3' and 5'
GCAGCTCGTTTTAGGCATTCGTTTAGCTTTTGGC 3') were used
to mutate codons Ile417 and Met421 to stop
codons (in bold). The resulting transformed BL21 (DE) cells
were screened for expression of the doubly truncated protein using
SDS-polyacrylamide gel electrophoresis. The mutation was confirmed by
sequencing the entire gene of a plasmid from a cell line that expressed
active TRH102-416; this was designated pEWOH
101
H.
Protein Expression and Purification--
Aliquots from frozen
cell stocks were plated (240 µl/liter of culture) on LB agar (100 µg/ml carbenicillin). After 9 h at 37 °C, the cells from two
plates were resuspended in 10 ml of LB broth and used to inoculate 1 liter of LB broth (100 µg/ml ampicillin). The culture was grown with
vigorous shaking at 37 °C for 2 h or until the cell density had
reached an A600 of 0.5. The culture flask was
then transferred to a second shaker at 20 °C and permitted to grow
for a further 30 min or until their cell density had reached an
A600 of 1.0. At this point
isopropyl-
-thiogalactoside was added to a final concentration of 0.1 mM. After 7.5 h the cells were harvested by
centrifugation at 4000 × g for 30 min and used
immediately for protein purification.
Unless otherwise stated all subsequent purification procedures were
undertaken at 4 °C. Cells were resuspended using 20 ml of 50 mM Tris-HCl, 100 mM
(NH4)2SO4, 2 mM
dithiothreitol, pH 8.0, per liter of culture and lysed with 6 bursts of
sonication for 40 s using a Branson sonicator fitted with a blunt
tungsten tip. The temperature of the solution was monitored to ensure
that it did not exceed 10 °C. The lysed cells were then centrifuged
at 11,200 × g for 30 min, and the pellet was
discarded. Polyethyleneimine was added to the supernatant to a final
concentration of 0.01%, and the mixture was allowed to stir for 10 min
and then centrifuged at 11,200 × g for 20 min. The
supernatant was loaded directly onto a Q-Sepharose column (22 cm3 liter
1 of culture) in the above buffer.
The initial fractions eluting from the Q-Sepharose column that
contained protein were combined. These were brought to 35%
(NH4)2SO4 saturation over a period
of 20 min and centrifuged at 7,800 × g for 20 min. The
resulting supernatant was then brought to 45%
(NH4)2SO4 saturation over a period
of 20 min and centrifuged at 7,800 × g for 20 min. The 45% supernatant was brought to 55%
(NH4)2SO4 saturation over a period
of 20 min and again centrifuged. The pellet obtained from this step was
redissolved in 50 mM MES, 200 mM
(NH4)2SO4, 10% glycerol, 2 mM dithiothreitol, 100 µM ferrous ammonium
sulfate, pH 7.0, using 25 ml per liter of initial culture. The
redissolved enzyme was applied to a ceramic hydroxyapatite column (11 cm3 per liter of initial culture) at a flow rate of 1 ml/min. The column was washed with approximately 2 column volumes of 50 mM MES, 200 mM
(NH4)2SO4, 10% glycerol, 2 mM dithiothreitol, 100 µM ferrous ammonium
sulfate, pH 7.0, and the protein was eluted with a linear gradient (18 column volumes) from this buffer to 300 mM sodium
phosphate, 10% glycerol, 2 mM dithiothreitol, 100 µM ferrous ammonium sulfate, pH 6.5. TRH102-416 typically eluted between 150 and 200 mM phosphate. Fractions containing TRH102-416
were pooled. Contaminating nucleic acids were removed by the further
addition of polyethyleneimine to 0.01% and stirring for 20 min. After
centrifugation at 11,200 × g for 20 min, the
supernatant was concentrated by the addition of
(NH4)2SO4 to 65% saturation over a
period of 20 min. This sample was then centrifuged at 7,800 × g for 15 min, and the supernatant was discarded. The white
protein pellet was redissolved in a minimum of 50 mM MES,
200 mM (NH4)2SO4, 10%
glycerol, 2 mM dithiothreitol, pH 7.0, and stored as small
aliquots (typically 200 µl of 200 µM enzyme) at
70 °C. When the enzyme was to be used for kinetic studies, the
storage buffer also contained 100 µM ferrous ammonium
sulfate. A typical yield from 1 liter of cells was 10 mg with a
specific activity of 0.6 µmol of hydroxytryptophan produced per
min/mg.
Ultracentrifugation--
Sedimentation equilibrium analyses were
carried out at 10 °C in a Beckman model XL-A ultracentrifuge.
Concentrated TRH102-416 was diluted to 5, 10, or 15 µM in 200 mM
(NH4)2SO4, 100 mM MES, pH 7.2. The system was assessed as having attained equilibrium when
scans at 280 nm taken at 2-h intervals were identical. The data used
for analysis were averages of 20 successive scans. The absorbance
values as a function of radial position were fit using Kaleidagraph
software to either Equation 1, which describes the equilibrium
sedimentation of a monomer, or Equation 2, which describes the
sedimentation of a self-associating species. The terms used in
Equations 1-3 are as follows: N, the stoichiometry of
association; M, the molecular mass of an enzyme monomer;
Ao, the absorbance at the reference radius
ro; Ka, the association
constant; C, the base-line offset;
, the
partial specific volume (calculated to be 0.723 from the amino acid
content of TRH102-416);
, the buffer density; and
,
the angular velocity in radians/s. The concentration of protein was
determined using an
280 value of 35.2 mM
1 cm
1 calculated by the
method of Pace et al. (22).
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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Enzyme Assays--
An HPLC-based assay for enzyme activity was
used for samples during purification. The 500-µl reaction mixture
contained enzyme, 0.2 mg/ml catalase, 200 µM tryptophan,
200 µM 6-MePH4, 100 µM ferrous
ammonium sulfate, 15 mM
-mercaptoethanol, 100 mM MES, pH 7.0. The reaction was initiated by the addition
of tetrahydropterin after equilibrating all other substituents at
37 °C for 1 min. Aliquots (100 µl) were withdrawn at times between
0 and 30 s and quenched into 10 µl of 40% trichloroacetic acid.
These samples were then centrifuged at 12,000 × g for
10 min and loaded onto a Rainin microsorb MV reverse phase C18 HPLC
column (50 × 4.6 mm inner diameter) using a 10-µl injection
loop. The mobile phase was 40 mM sodium acetate, 5%
acetonitrile, pH 3.5, at flow rate of 1 ml/min. 5-Hydroxytryptophan and
tryptophan were detected using a Waters 470 fluorescence detector with
an excitation
of 290 nm and an emission
of 340 nm. Under these
conditions 5-hydroxytryptophan and tryptophan had retention times of
1.7 and 3.9 min, respectively. When phenylalanine was used as a
substrate, tyrosine was detected with an excitation
of 275 nm and
an emission
of 310 nm, using 40 mM sodium acetate, pH
3.5, as the mobile phase. Similarly, the product of tyrosine
hydroxylation, 3,4-dihydroxyphenylalanine, could be detected with an
excitation
of 280 nm and an emission
of 310 nm in the same
mobile phase.
Steady state kinetic measurements with tryptophan as substrate were
made using an Applied Photophysics stopped-flow apparatus operating in
the fluorescence mode. TRH102-416 (typically 2-10
µM) in air-saturated 200 mM
(NH4)2SO4, 100 mM MES,
100 µM ferrous ammonium sulfate, 25 µg/ml catalase, pH
7.0, was rapidly mixed with aerobic solutions containing varied
concentrations of tetrahydropterin and tryptophan in 10 mM
HCl and 12 mM dithiothreitol at 15 °C. The excitation
wavelength was 300 nm; all emitted light which passed a 320-nm
wavelength cut-off filter perpendicular to the light source was
collected. Under such conditions, the formation of 5-hydroxytryptophan
could be monitored independently of the substrate or the formation of
the fluorescent 7,8-dihydropterin. The amount of 5-hydroxytryptophan
generated was quantified by comparing the fluorescence yield to that of
5-hydroxytryptophan standards measured on the same instrument in the
presence of comparable quantities of other substrates. The
concentration dependence data were fit to Equations 4 and 5. Equation 5
was used when substrate inhibition was observed; Kai
is the inhibition constant for the substrate.
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(Eq. 4)
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(Eq. 5)
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The metal requirement of TRH102-416 was determined
using sequential stopped-flow spectrophotometry. The apoenzyme (10 µM, 2.5 µM final concentration) in 100 mM MES, 200 mM
(NH4)2SO4, pH 7.0, was first mixed
with 6-MePH4 (400 µM, 100 µM
final concentration) and tryptophan (200 µM, 50 µM final concentration). After 1 s this was mixed
with another solution containing 100 µM metal ion (50 µM final concentration). The second mixing step initiated data collection.
A modified version of the assay of Shiman et al. (23) was
used to measure tyrosine formation from phenylalanine. Enzyme (typically 2-10 µM) in air-saturated 100 mM
MES, 200 mM (NH4)2SO4, 100 µM ferrous ammonium sulfate, 25 µg/ml catalase, pH
7.0, was mixed with aerobic solutions containing varied concentrations of tetrahydropterin and phenylalanine in 10 mM HCl and 12 mM dithiothreitol at 15 °C.
A variation of the method of Fitzpatrick (12) was used to assay
tyrosine hydroxylation. The conditions were 100 mM MES, 200 mM (NH4)2SO4, 100 µM ferrous ammonium sulfate, 25 µg/ml catalase, pH 7.0, with varied concentrations of 3,5-[3H]tyrosine and
tetrahydropterin. The reaction was carried out for 1 min at
15 °C.
Pterin Products and Stoichiometry--
Enzyme (10 µM final) was mixed with tryptophan (200 µM), phenylalanine (200 µM), or tyrosine
(650 µM) in 100 mM MES, pH 7.2, 200 mM (NH4)2SO4, 100 µM ferrous ammonium sulfate, at 15 °C, in a 4-mm path
length quartz cuvette. After recording a base line with this solution,
the reaction was initiated by the addition of either
6-MePH4 or BH4 to 100 µM, and
spectra were recorded every 5 s for 400 s. The amount of
hydroxylated amino acid produced was then determined by HPLC. The
spectra generated during the reaction were analyzed globally using the
program Specfit (Spectrum Software Associates) to determine the spectra
of the pterin products.
Iron Determination--
The iron content of the enzyme was
determined by atomic absorption spectroscopy, using a slight variation
of the method of Ramsey et al. (24). Samples were dialyzed
into 100 mM MES, 200 mM
(NH4)2SO4, pH 7.0, and then diluted
5-fold in 2.5 M nitric acid. After 30 min on ice the
samples were diluted 10-fold with water and centrifuged at 12,000 × g for 10 min. Aliquots of the supernatant were analyzed
for iron using a Perkin-Elmer model 2380 atomic absorption
spectrophotometer equipped with a graphite furnace.
 |
RESULTS |
Expression--
The T7 polymerase-based pET expression system of
Studier (25) was used to express wild type TRH and two truncated mutant proteins. The wild type TRH was expressed as less than 1% of the total
cell protein. This level of expression was unaffected by temperature
over the range 20-37 °C (data not shown). At 20 °C, the TRH
activity reached a maximum level of approximately 4 nmol/min/ml culture
7 h after induction (Fig.
1A). All of the wild type TRH was soluble. In contrast, the protein lacking the amino-terminal regulatory domain, TRH102-444, was expressed at much
higher levels. Based upon SDS-polyacrylamide gel electrophoresis of
cell lysates after induction, TRH102-444 was expressed as
30-35% of the total cell protein under all conditions tested.
However, when cells were grown at 37 °C, 95% of the
TRH102-416 was found in inclusion bodies. The fraction of
soluble TRH102-444 could be increased by decreasing the
growth temperature to 17 °C, but the greatest activity found under
any condition with this protein was only 40% that observed with the
wild type protein (see Fig. 1A). Moreover, during attempts
to purify TRH102-444, the enzyme showed a pronounced
tendency to lose activity and precipitate.

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Fig. 1.
Expression and purification of
TRH102-416. A, catalytic activity (filled
symbols) and cell density (open symbols) of BL(DE3)
cells expressing wild type TRH ( , ), TRH102-444
( , ), or TRH102-416 ( , ).
Isopropyl- -thiogalactoside was added at 2 h. B,
SDS-polyacrylamide gel electrophoresis of fractions obtained in the
purification of TRH102-416. Lane 1, molecular
weight markers: rabbit myosin, 205,000; E. coli
-galactosidase, 116,000; rabbit phosphorylase b, 97,000;
bovine albumin, 66,000; ovalbumin, 45,000; glyceraldehyde-3-phosphate
dehydrogenase, 36,000; carbonic anhydrase, 29,000; bovine trypsinogen,
24,000. Lane 2, cell lysate. Lane 3, 45-55%
(NH4)2SO4 pellet. Lane
4, ceramic hydroxyapatite column pool.
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Although the absolute levels of expression seen with
TRH102-444 were sufficient, the insolubility of the
protein and the low activity suggested that it was not folding
properly. TRH is believed to be held together as tetramer by
hydrophobic helices at the carboxyl termini of each monomer (6, 8, 16,
26, 27), similar to TYH (13, 18). Given the tetrameric nature of TRH,
it was considered possible that the presence of even a single unfolded
subunit within the tetramer would be sufficient to render the entire
tetramer unstable. Since the interface helix is not required for
activity in these hydroxylases (13, 15, 27), a mutant protein lacking
both the regulatory domain and the carboxyl-terminal helix was
examined. This form of TRH, TRH102-416, was expressed at a
level similar to that observed with TRH102-444. However,
based on SDS-polyacrylamide gel electrophoresis and enzyme activity,
80% of the protein was soluble, so that the active, soluble
TRH102-416 was 25-30% of the total cell protein. The
amount of enzyme activity produced as TRH102-416 was approximately 10 times that observed with the wild type enzyme and 25 times that obtained with TRH102-444 (Fig.
1A). Consequently, TRH102-416 was selected for
further characterization.
Purification--
TRH102-416 could be purified in
three steps, a Q-Sepharose column, ammonium sulfate fractionation, and
a hydroxyapatite column (Fig. 1B). TRH102-416
showed marked instability at low ionic strength. Maintaining the enzyme
in a minimum of 100 mM
(NH4)2SO4 during purification
greatly enhanced recovery from each chromatographic step. The enzyme
stability was further enhanced by ferrous iron and dithiothreitol. The
enzyme could be stored at
70 °C indefinitely without loss of
activity. Because of the improved stability which resulted, the enzyme
was typically stored in the presence of 100 µM ferrous
ammonium sulfate. If ferrous ammonium sulfate was omitted from
purification buffers, the resulting enzyme contained no detectable iron
(<0.05 atom/monomer).
Ultracentrifugation--
Since TRH102-416 no longer
contains the helix that is proposed to be necessary for
oligomerization, the mutant protein should be a monomer. Equilibrium
ultracentrifugation was used to analyze its quaternary structure. Data
were collected using initial protein concentrations of 5-15
µM and rotor speeds of 16,000, 19,000, and 22,000 rpm.
When the data were analyzed assuming that a single monomeric species
was present, the average molecular weight over all conditions was
42,400 ± 4381, compared with a molecular weight of 36,319 calculated from the DNA sequence. These results suggested that species
larger than a monomer were present. To examine this possibility, the
data were fit to models describing monomer-dimer, monomer-trimer,
monomer-tetramer, monomer-hexamer, and monomer-octamer equilibria. In
each case the molecular weight was fixed at 36,319. The data were best
fit using a monomer-tetramer model (Table
I). No improvements in the quality of the
fits were seen if species larger than a tetramer were considered. The
average Ka value obtained by fitting each data set
to a model describing a monomer-tetramer equilibrium was 1.33 × 1013 M
3. Fig.
2 shows representative fits to this model
of data obtained at three enzyme concentrations using the average
Ka value and a monomer molecular weight of 36,319. Based on this association constant, TRH102-416 is 50%
monomer at a protein concentration of 42 µM.
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Table I
Ultracentrifugation analyses of the quaternary structure of
TRH102-416
200 mM (NH4)2SO4, 100 mM MES, pH 7.2, 10 °C. The system was assessed as having
attained equilibrium when scans at 280 nm taken at 2-h intervals were
identical.
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Fig. 2.
Analytical ultracentrifugation of
TRH102-416 at 22,000 rpm. The initial concentrations
of enzyme before centrifugation began were 5 µM ( ---),
10 µM ( - - -), or 15 µM
( - - -). The lines represent fits of the data to a
monomer-tetramer equilibrium with a Ka value of
1.3 × 1013 M 3 using
Equation 2.
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Hydroxypterin Formation--
With tyrosine hydroxylase and
phenylalanine hydroxylase, the initial pterin product of catalysis is a
4a-hydroxypterin (28-30). Although it is assumed that this
is also a product with tryptophan hydroxylase, as shown in
Scheme 1, formation of a hydroxypterin by
tryptophan hydroxylase has not been demonstrated directly. Although
hydroxypterins are not stable in solution for extended periods,
they can be observed spectrally if the enzyme concentration is high
enough to rapidly generate micromolar levels prior to hydrolysis.
Consequently, high concentrations of TRH102-416 (10 µM) were used to consume 100 µM
tetrahydrobiopterin in the presence of excess tryptophan or
phenylalanine. Near ultraviolet absorbance spectra of the reaction were
collected every 5 s using a diode array spectrophotometer. The
formation of the hydroxypterin was clearly detectable at 246 nm, where
its absorbance is maximal, when either tryptophan or phenylalanine was
the amino acid substrate (Fig. 3). After
the spectral changes were complete, the amount of hydroxylated amino
acid produced was determined by HPLC. With tryptophan as substrate 103 nmol of hydroxytryptophan were produced after complete oxidation of 100 nmol of tetrahydrobiopterin. Similarly, oxidation of 100 nmol of
tetrahydrobiopterin in the presence of phenylalanine produced 99 nmol
of tyrosine. Similar results were obtained with 6-MePH4
(Table II). However when tyrosine was
used as a substrate only 1.2 nmol of 3,4-dihydroxyphenylalanine were produced upon the oxidation of 100 nmol of tetrahydrobiopterin.

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Fig. 3.
Formation of 4a-hydroxypterin
during turnover by TRH102-416. A, spectra at
5-s intervals during the turnover of 134 µM
BH4 by 10 µM TRH102-416 with 200 µM tryptophan in 100 mM MES, 200 mM (NH4)2SO4, 100 µM ferrous ammonium sulfate, and 320 µM
oxygen, pH 7.0, at 15 °C. B, spectra accumulated at 5-s
intervals during the turnover of 133 µM BH4
by 10 µM TRH102-416 with 200 µM phenylalanine in 100 mM MES, 200 mM (NH4)2SO4, 100 µM ferrous ammonium sulfate, and 320 µM
oxygen, pH 7.0, at 15 °C. C, the spectra of
BH4 (- - -) and of 4a-hydroxy-BH3
(---) determined from the reaction depicted in B.
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The sequential spectra collected during turnover were fit globally to
the model in Scheme 2 to determine the
spectra of the individual pterin species produced. The data were well
fit by such a model (results not shown). The spectrum of the
4a-hydroxybiopterin determined by this method agreed well
with previously published spectra (31), with an
246
value of 18.1 mM
1 cm
1 (Fig. 3).
The spectra of the subsequently formed pterin species agreed with
previously described spectra of the quinonoid dihydrobiopterin and
7,8-dihydrobiopterin (31, 32).
Steady State Kinetic Analyses for
TRH102-416--
Continuous assays were developed as a
consequence of the substantial increase in activity of
TRH102-416 compared with TRH from other sources. The assay
for hydroxylation of tryptophan followed the increased fluorescence of
the product at wavelengths greater than 320 nm. Selectively exciting
the reaction at 300 nm minimized the emission contributions from the
substrate and fluorescent pterin products. With the alternate substrate
phenylalanine, the accumulation of tyrosine could be observed as an
absorbance increase at 275 nm.
Tryptophan, phenylalanine, and tyrosine were examined as substrates for
TRH102-416. Both tryptophan and phenylalanine were
hydroxylated efficiently, producing exclusively 5-hydroxytryptophan and
4-hydroxyphenylalanine, respectively. Under the experimental conditions, no production from phenylalanine of
3-hydroxyphenylalanine or 3,4-dihydroxyphenylalanine could be detected
by HPLC. Very small quantities of 3,4-dihydroxyphenylalanine formed
from tyrosine could be detected. The hydroxylation rate of tyrosine by
TRH102-416 under the conditions of the experiment was at
least 5000-fold slower than that observed with tryptophan and
phenylalanine. For this reason steady state kinetic parameters were
determined only with tryptophan and phenylalanine using
6-MePH4 and BH4. Significant substrate
inhibition was seen when either amino acid was varied at a fixed
concentration of either BH4 or 6-MePH4 (Fig.
4). Substrate inhibition was also seen
when either tetrahydropterin was varied with tryptophan, but little or
no inhibition was seen with either tetrahydropterin when phenylalanine
was the fixed substrate (Fig. 4). Table
III summarizes the kinetic
parameters.

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Fig. 4.
Steady state kinetic analyses for
TRH102-416. A, initial rates of tryptophan
hydroxylation as a function of tryptophan concentration with 100 µM 6-MePH4 ( ) or 150 µM
BH4 ( ). The lines are from fits of the data
to Equation 5. B, initial rates of phenylalanine
hydroxylation as a function of phenylalanine concentration with 100 µM 6-MePH4 ( ) or 150 µM
BH4 ( ). The lines are from fits of the data
to Equation 5. C, initial rates of amino acid hydroxylation
as a function of tetrahydrobiopterin concentration with 100 µM tryptophan ( ) or 100 µM phenylalanine
( ). The lines are from fits of the data to equation 4 for
phenylalanine and to Equation 5 for tryptophan. D, initial
rates of amino acid hydroxylation as a function of 6-MePH4
concentration with 100 µM tryptophan ( ) or 100 µM phenylalanine ( ). The lines are from
fits of the data to Equation 5.
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Table III
Steady state kinetic parameters for TRH102-416
Conditions were as follows: 50 mM MES, 100 mM
(NH4)2SO4, 6 mM dithiothreitol, pH
7.0, 15 °C.
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The Metal Requirement of TRH102-416--
Both
tyrosine hydroxylase and phenylalanine hydroxylase have been shown to
be iron-containing enzymes (33-35), so that tryptophan hydroxylase is
also assumed to require iron for activity. If no iron was included in
the buffer when purifying TRH102-416, the resulting
protein contained no significant iron. Apoenzyme prepared in this
fashion was used to determine the metal requirement of tryptophan
hydroxylase. The assays were done at high (>2 µM) enzyme
concentrations to avoid ambiguities due to the presence of
contaminating metals. The apoenzyme was mixed with tryptophan and
6-MePH4 and then 1 s later with individual metals at a
final concentration of 50 µM. The formation of
hydroxytryptophan was followed by fluorescence. Representative kinetic
traces are shown in Fig. 5. No activity
was observed in the absence of added metal. In the presence of 50 µM ferrous iron there was a constant rate of product
formation beginning almost immediately after mixing. The same rate was
seen with 10 µM ferrous iron (results not shown). In
contrast, in the presence of an equal concentration of ferric iron, a
significant lag was seen before the activity reached the same level as
was seen with ferrous iron. The enzyme was not active with copper or
cobalt.

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Fig. 5.
The metal requirement of
TRH102-416. Traces show the fluorescence changes due
to 5-hydroxytryptophan formation upon mixing
apo-TRH102-416 with the indicated metals at a
concentration of 50 µM in the presence of
6-MePH4 and tryptophan at 15 °C.
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DISCUSSION |
The three tetrahydropterin-dependent hydroxylases,
TYH, PAH, and TRH, constitute a small family of proteins that catalyze the hydroxylation of aromatic amino acids. Because of the availability of PAH from liver, studies of this enzyme have the most extensive history (36). More recently, the availability of recombinant TYH has
resulted in a much greater understanding of that enzyme (10). In
contrast, understanding the structure and mechanism of tryptophan
hydroxylase has made little progress. Natural sources such as brain
have proved to contain too little enzyme for useful purification,
resulting in very low amounts of protein with low specific activities
(1, 2, 4). A number of groups have described preparations of TRH from
recombinant sources, but these typically involved enzyme of low or
indeterminate specific activity (16, 37, 38). The limited amounts of
material available have severely restricted analyses of the mechanism
of this important enzyme.
Sequence comparisons of all three hydroxylases routinely show that the
carboxyl-terminal 340 amino acids form a homologous region, whereas the
amino-terminal sequences diverge widely (20). Deletion mutageneses of
all three enzymes have defined the catalytic core which contains the
residues required for catalysis. PAH lacking the amino-terminal 141 amino acid residues and the carboxyl-terminal 43 residues is reported
to retain activity (15). Similar results have been reported for TYH
(13, 14), whereas TRH is reported to retain some activity if as many as
106 residues are deleted from the amino terminus and as many as 19 residues are deleted from the carboxyl terminus (9, 39). Based upon
such results, the amino-terminal portions of these proteins are
generally accepted to be regulatory domains, in that they contain
phosphorylation sites and are required for allosteric properties (37,
40-42). The 42 carboxyl-terminal residues of each monomer are
responsible for tetramer formation, primarily due to the presence of a
24-residue helix at the end (13, 18). The remaining 300 residues form the catalytic domains of each hydroxylase. Removal of the regulatory domains from either TYH or PAH has only subtle effects on the substrate
specificities or catalytic rates (17).
In our hands, expression of the wild type rabbit TRH resulted in low
levels of expression, consistent with the observations of others (38).
Although removal of the regulatory domain resulted in a significant
increase in the level of expression of TRH102-444, this
form of the protein was only slightly soluble. It was only upon removal
of the long helix in the tetramer interface that high levels of soluble
active enzyme were obtained. A possible reason for this increase in
solubility of TRH102-416, the protein lacking both the
regulatory domain and the tetramerization helix, is that tetramers of
TRH102-444 contain mixtures of correctly folded and
incorrectly folded monomers. Any improperly folded subunit may render
an entire tetramer unstable. Even if not all of the monomeric
TRH102-416 is correctly folded, any improperly folded
subunits would not be expected to affect the stability of other
monomers. Irrespective of whether this is the correct reason for its
increased solubility, TRH102-416 is expressed at
sufficiently high levels for mechanistic studies.
Wild type TRH is a tetramer (1, 4). In contrast,
TRH102-416 is monomeric at and above concentrations
typically encountered in kinetic experiments and only forms oligomers
at a relatively high
concentration.2 This is the
result expected upon removal of the intersubunit helix. There are
clearly still some interactions among the monomers in
TRH102-416, despite the lack of this helix. The structure of TYH shows that the tetramerization domain contains the carboxyl 42 residues, which include the intersubunit helix (18). In addition, there
are other interactions across dimer interfaces. Similar interactions in
TRH are presumably the reason for the weak formation of tetramers.
TRH102-416 clearly requires ferrous iron for activity, as
do both TYH and PAH (11, 12, 43). Although TRH102-416 is
active with ferric iron, there is a significant lag in formation of
hydroxytryptophan in the presence of this metal. Both TYH and PAH are
routinely found to have the active site iron in the ferric form when
purified (44, 45). The iron must be reduced to the ferrous form for
catalysis; tetrahydrobiopterin appears to be the physiological
reductant (11, 24, 43). The lag seen in the formation of
hydroxytryptophan by TRH102-416 in the presence of ferric
iron is consistent with a similar phenomenon occurring with this
protein. The lag would be due to the relatively slow reduction of the
ferric enzyme by the tetrahydropterin.
Based upon the precedents with TYH and PAH (28-30), it was expected
that the initial pterin product with TRH would be the 4-hydroxypterin. The high levels of TRH102-416 have made it possible to
demonstrate this directly for the first time, establishing the reaction
shown in Scheme 1 for TRH. Moreover, the stoichiometry of one
tetrahydropterin consumed per hydroxylated amino acid produced shown in
Scheme 1 has been established for both the physiological substrate
BH4 and the synthetic substrate 6-MePH4 with
both tryptophan and phenylalanine as the amino acid substrate. Thus,
based upon the degree of coupling of tetrahydropterin consumption to
tyrosine formation, phenylalanine is as good a substrate as tryptophan
for TRH102-416. With both TYH and PAH it has commonly been
observed that use of nonphysiological substrates results in an excess
of tetrahydropterin consumed over amino acid hydroxylated (36, 46, 47).
Indeed this is also the case when tyrosine is the substrate for
TRH102-416 where there is an 80-fold greater
tetrahydropterin oxidation than amino acid hydroxylation.
The steady state kinetic analyses presented here for
TRH102-416 provide insight into the substrate specificity
of TRH and allow comparison with PAH and TYH. Qualitatively, tyrosine is a poor substrate for TRH102-416, and both tryptophan and phenylalanine are good substrates. Indeed, given that the kinetic
parameters in Table III could not be determined at saturating concentrations of the nonvaried substrates due to substrate inhibition, the kinetic parameters for tryptophan and phenylalanine are probably not significantly different. These results can be compared with the
substrate specificities of TYH and PAH. It is best to use results
obtained with the comparable catalytic domains of the latter enzymes
because of the complications caused by the need for prior activation of
wild type PAH. Other than release from cooperative substrate activation
of PAH, removal of the regulatory domains of PAH and TYH does not
affect the ability of these enzymes to hydroxylate the other aromatic
amino acids (17). TYH will hydroxylate phenylalanine (48, 49).
Whereas the rate of tetrahydropterin oxidation by TYH in the presence
of phenylalanine is comparable to that seen in the presence of
tyrosine, only a fraction of the reducing equivalents are used to form
tyrosine (17, 49). This is in contrast to the situation with
TRH102-416, in which tetrahydropterin oxidation and amino
acid hydroxylation are stoichiometric. Tryptophan is also a substrate
for TYH, but with a Vmax value only 20% that
seen with tyrosine and with a Km value 20-fold
higher (50). Thus, tyrosine is clearly the preferred substrate for TYH.
PAH is unable to hydroxylate tyrosine, although tyrosine does stimulate
a low rate of tetrahydropterin oxidation (17, 49, 51). In contrast, PAH
is able to hydroxylate tryptophan (42, 52) but with a
Km value a 1000-fold higher and a
Vmax value one-tenth that of phenylalanine.
Thus, PAH strongly prefers phenylalanine over the other two amino acids
as a substrate. While TRH102-416 resembles PAH
qualitatively in its ability to hydroxylate only phenylalanine and
tryptophan, TRH102-416 shows no discernible preference for
its physiological substrate. It is not clear whether there is
physiological relevance to this lack of specificity.
In conclusion, the goal of the work presented here was to obtain a form
of TRH that would permit study of catalytic properties. The results
indicate that rabbit TRH102-416 is a stable, highly active
form of TRH that can be expressed to very high levels in E. coli. This mutant enzyme clearly appears to be valid for mechanistic studies of wild type TRH.