From the Biochemisches Institut der Universität Zürich, CH-8057 Zürich, Switzerland
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ABSTRACT |
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Tyrosine phenol-lyase (TPL), which catalyzes the
The pyridoxal 5'-phosphate-dependent enzymes
(B6 enzymes) catalyze a wide variety of reactions in the
metabolism of amino acids (1). A comparison of amino acid sequences has
shown that the majority of B6 enzymes belong to the large
-elimination reaction of L-tyrosine, and aspartate
aminotransferase (AspAT), which catalyzes the reversible transfer of an
amino group from dicarboxylic amino acids to oxo acids, both belong to
the
-family of vitamin B6-dependent enzymes.
To switch the substrate specificity of TPL from L-tyrosine
to dicarboxylic amino acids, two amino acid residues of AspAT, thought
to be important for the recognition of dicarboxylic substrates, were
grafted into the active site of TPL. Homology modeling and molecular
dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which
binds the distal carboxylate group of substrates and is conserved among
all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in
AspAT, which interacts with the phosphate group of the coenzyme. The
double mutation R100T/V283R of TPL increased the
-elimination
activity toward dicarboxylic amino acids at least 104-fold.
Dicarboxylic amino acids (L-aspartate,
L-glutamate, and L-2-aminoadipate) were
degraded to pyruvate, ammonia, and the respective monocarboxylic acids,
e.g. formate in the case of L-aspartate. The
activity toward L-aspartate (kcat = 0.21 s
1) was two times higher than that toward
L-tyrosine.
-Elimination and transamination as a minor
side reaction (kcat = 0.001 s
1)
were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in
its reaction specificity. Dicarboxylic amino acid
-lyase is an
enzyme not found in nature.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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-superfamily of homologous B6 enzymes (2, 3).
Tyrosine phenol-lyase (TPL)1
of Citrobacter freundii is a member of the
-family. It
catalyzes the
-elimination of L-tyrosine to produce
phenol, pyruvate, and ammonium (Equation 1).
A number of amino acids with suitable leaving groups on C
(Eq. 1)
, such
as L-serine and O-acyl-L-serines
(4), L-cysteine, S-alkyl-L-cysteines (4, 5), and 3-chloro-L-alanine, are also substrates for
-elimination. Moreover, TPL has been found to catalyze markedly slower side reactions, i.e.
-replacement reactions (6,
7), racemization of alanine (8, 9), as well as transamination reactions
of its substrates L-tyrosine, L-serine, and of
the competitive inhibitors L-alanine,
L-phenylalanine, and L-m-tyrosine
(10).
X-ray crystallographic structure analysis has shown the folding pattern
of the polypeptide chain of tetrameric TPL from C. freundii
to be similar to that of dimeric aspartate aminotransferase (AspAT)
(11), which, like TPL, is a member of the -family of pyridoxal
5'-phosphate (PLP)-dependent enzymes. Despite their similarity in secondary and tertiary structure, the two enzymes show
only low sequence identity, e.g. 23% between TPL of
C. freundii and AspAT of Escherichia coli. AspAT
catalyzes the reversible transamination reaction of the dicarboxylic
L-amino acids aspartate and glutamate with the cognate
2-oxo acids 2-oxoglutarate and oxalacetate.
The structures of the active sites of TPL and AspAT are similar; most
of the residues that participate in the binding of the coenzyme and the
-carboxylate group of the substrate in AspAT (12) are conserved in
the structure of TPL (13). Obviously, these two homologous enzymes use
the same protein scaffold to catalyze different reactions with
different substrates. Thus, alteration of the specificity of a given
enzyme by substitution of a limited number of critical amino acid
residues seems feasible. Alignments of amino acid sequences of
homologous enzymes may be used to identify the residues underlying the
differences in their reaction and substrate specificity. Substitution
of the residues to which the substrate binds has proven successful in
changing the substrate specificity of several enzymes without
destroying their catalytic apparatus (14-21).
This paper reports a homology modeling approach that, together with
information obtained from structural and mechanistic studies of AspAT,
was used to redesign the substrate specificity of TPL in favor of
dicarboxylic amino acids.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction and Mutagenesis-- Plasmid pTZTPL (22) containing the entire coding sequence of C. freundii tyrosine phenol-lyase was used as template for in vitro mutagenesis. The tpl gene was amplified by the polymerase chain reaction using the following two synthetic oligonucleotides as primers: 5'-CGCGCGTCGACATAATTATTATTTAGTGATGATGATGATGATGGATATAGTCAAAGCG-3' and 5'-GCGGAGATCTAACTCACTG-3'. The first oligonucleotide hybridizes to the 5' part of the tpl gene and contains six histidine codons (italics), in frame, just before the stop codon and a new SalI site (underlined). The second oligonucleotide hybridizes to the unique BglII site (underlined) in the tpl gene upstream to the transcriptional start point. The resulting 1.9-kilobase pair polymerase chain reaction product was cut with BglII and SalI and subcloned into the BamHI-SalI sites of the expression vector pTZ18U (Bio-Rad) to generate pTZTPL-His.
The mutants were prepared by polymerase chain reaction from pTZTPL-His using the QuikChangeTM Site-directed Mutagenesis Kit from Stratagene and the following primer pairs: R100Ta, 5'-CCTACTCACCAGGGGACCGGCGCAGAAAACCTG-3'; R100Tb, 5'-CAGGTTTTCTGCGCCGGTCCCCTGGTGAGTAGG-3'; V283Ra, 5'-CTTCGTACACACGGACTAAC-3'; and V283Rb, 5'-GTTAGTCCGTGTGTACGAAG-3'. The insertion of the histidine codons and the mutations was verified by cycle sequencing (Sequi Therm Long-Read Cycle Sequencing Kit-LC, Epicentre Technologies) with fluorescent primers using a DNA sequencer (LI-COR).
Expression and Purification--
E. coli SVS370 cells
were used as host for the pTZTPL-His and the mutant plasmids. The
cells, grown as described previously (22), were thawed and suspended in
5 ml of Buffer A (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, 1 mM
phenylmethylsulfonyl fluoride, 5 mM 2-mercaptoethanol, 0.1 mM PLP, pH 8.0) per gram of wet weight. The cells were
disrupted by three passages through a French press. Cell debris was
removed by centrifugation at 25,000 × g at 4 °C for
30 min. The supernatant was passed through a 0.22-µm filter and
directly applied onto a 13 × 1-cm column containing 2-3 ml of
nickel-nitrilotriacetic acid-agarose (Qiagen) equilibrated with Buffer
A. The column was washed with Buffer A containing 20 mM
imidazole until A280 of the flow-through
solution was below 0.01. The TPL protein was then eluted with a 30-ml
gradient from 20 to 250 mM imidazole in Buffer A. The
pooled TPL fractions were dialyzed extensively against 0.1 M potassium phosphate, pH 7.0, containing 0.1 mM PLP, 1 mM EDTA, and 5 mM
2-mercaptoethanol. Purified wild-type and mutant TPLs were stable at
least for 1 year when stored at 70 °C in the same buffer at a
concentration of 2-5 mg/ml. All preparations were pure as indicated by
SDS-polyacrylamide gel electrophoresis (10-15% PHAST-gel from
Amersham Pharmacia Biotech).
Protein Determination--
The concentration of purified TPLs
was determined photometrically
(E2781% = 8.37; Ref. 5) assuming
a subunit molecular mass of 52.3 kDa (13) which takes into account the
molecular mass of the His6 tag (0.84 kDa). The PLP content
of the enzymes was determined from the absorption spectrum of the
enzyme in 0.1 M NaOH, assuming 388 = 6600 M
1 cm
1 (23).
Absorption Spectra of Tyrosine Phenol-lyase-- Prior to recording absorption spectra, the stock enzyme was incubated with 0.5 mM PLP for 1 h at 30 °C and then separated from excess PLP on a short desalting column (NAPTM 5, Amersham Pharmacia Biotech) equilibrated with 50 mM potassium phosphate, pH 8.0. Absorption spectra were measured with a 8453 UV-visible diode-array spectrophotometer from Hewlett-Packard.
Measurement of -Elimination Activity--
The activity of the
TPLs toward various amino acid substrates was measured using the
coupled assay with lactate dehydrogenase and NADH previously described
for tryptophan indole-lyase (24). The standard assay mixture contained
50 mM potassium phosphate, pH 8.0, 5 mM
2-mercaptoethanol, 50 µM PLP, 0.2 mM NADH, 24 units of lactate dehydrogenase from bovine heart (Sigma), and varying concentrations of amino acid substrate in a final volume of 1 ml at
25 °C. The reaction was initiated by the addition of TPL and
followed by the decrease in absorbance at 340 nm. Steady-state kinetic
values of kcat and Km were
obtained by fitting the data to the Michaelis-Menten equation using
ORIGIN software (Microcal Software).
Measurement of Transaminase and Racemase Activity-- Mutant TPLs and wild-type enzyme were incubated in 50 mM potassium phosphate, pH 8.0, with different amino acids as substrates. Samples were withdrawn at different times and immediately deproteinized with 1 M perchloric acid (25). After derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (26), the reaction samples were loaded onto a reverse-phase high pressure liquid chromatography column (Aquapore RP-300; 250 × 4.6-mm). Both pyridoxamine 5'-phosphate (PMP), produced by the single turnover half-reaction of transamination, and the products of racemization can be separated and detected photometrically at 340 nm by this sensitive assay (27). Alternatively, the increase in absorbance at 325 nm was used to follow the production of PMP.
Thin Layer Chromatography-- Protein was eliminated from the reaction mixture prior to chromatography by precipitation with 1 M perchloric acid (25). Thin layer chromatography was performed on pre-coated silica gel plates SIL G-25 from Macherey-Nagel in n-pentyl formate/chloroform/formic acid (20:70:10, v/v). A slightly alkaline solution of bromcresol green (0.02% in ethanol) was used to develop the chromatogram. The acids appeared as yellow spots on a blue background (28).
Formate Dehydrogenase Assay-- Formic acid was determined with the formate dehydrogenase assay. A kit from Boehringer Mannheim was used according to the supplier's protocol. Briefly, mutant TPLs were incubated at 25 °C in 50 mM potassium phosphate, pH 8.0, with L-aspartate as substrate. Samples were withdrawn at different times and immediately deproteinized with perchloric acid. Formate was quantitated by the increase in absorbance at 340 nm due to NAD+ reduction.
Molecular Modeling and Dynamics Simulations--
The crystal
structure of the holoenzyme complex with the substrate analog
3-(4-hydroxyphenyl) propionic acid (Brookhaven Protein Data Bank, code
2TPL) was used as parent structure. The substrate analog was replaced
by L-tyrosine, and the external aldimine form 1 (Scheme 1) was created by introducing a
double bond between C4' and the nitrogen atom of the substrate. Removal
of the C-hydrogen, change of the hybridization of the C
atom from
sp3 to sp2, and
subsequent minimization led to the quinonoid intermediate 2.
Molecular dynamics simulations of this intermediate were performed
using the Discover program (Molecular Simulations) with the consistent
valence force field. The cell multipole method was used instead of a
cut-off for the nonbonded interactions. The temperature was set to 400 K. All hydrogen atoms and explicit water molecules were included in the
simulations with time steps of 1 fs. At the beginning, the whole system
was minimized for 2000 steps. The outer shell was then kept fixed, and
another 2000 steps of minimization were applied. This was followed by a
molecular dynamics simulation, which was initialized at 400 K for 1000 fs. After this initialization, the outer shell was again kept fixed. The simulation was continued for a total time of 20 ps. Every 100 fs
the potential energy was analyzed. Within each picosecond, only the
structure with the lowest potential energy was stored, resulting in a
total of 20 low energy structures. All these 20 structures were then
minimized for 2500 steps. The resulting minimized structures were found
to be generally quite similar, and one of these corresponding to the
average structure was chosen as starting point for all further
simulations. The modeled structure of the wild-type enzyme with
L-aspartate as substrate was obtained by replacement of
L-tyrosine and applying the same minimization-dynamics procedure as for the wild-type structure with L-tyrosine as
substrate. To model the double mutant enzyme, we replaced Arg-100 by a
threonine and Val-283 by an arginine residue and applied again the
minimization-dynamics procedure.
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RESULTS |
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Design Strategy-- In order to change the substrate specificity of TPL in favor of dicarboxylic amino acids, we compared TPL with AspAT using homology modeling and molecular dynamic simulations. The specificity of AspAT for dicarboxylic amino acids and oxo acids seems to be based primarily on the salt bridge-hydrogen bond interaction of the side chain of Arg-292 (of the adjacent subunit) with the distal carboxylate group of these substrates (12). In agreement with this notion, Arg-292 is conserved among all AspATs (29). Since the sequence identity between AspAT and TPL is too low (23%) to allow the use of standard alignment algorithms, comparison of their three-dimensional structures (13, 30) by superposition (Fig. 1) and with the program DALI (Fig. 2) was used to identify in TPL the residue corresponding to Arg-292 in AspAT. Val-283 in TPL seems to occupy the same position as Arg-292 in AspAT. Another significant difference in the active sites of these two enzymes is the replacement of a residue interacting with the phosphate group of the coenzyme. Arg-100 in TPL apparently corresponds to Thr-109 in AspAT which is also conserved among all AspATs (Figs. 1 and 2; Ref. 29).
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Molecular modeling showed that the quinonoid adduct of
L-aspartate and PLP can be sterically accommodated in the
active site of wild-type TPL (Fig. 3).
However, the orientation of the leaving group of the substrate relative
to the planar coenzyme-substrate adduct did not appear to be optimum
for a -elimination reaction. Positively charged Arg-100 in the
hydrophobic active site of TPL interacted with the distal carboxylate
group of dicarboxylic substrates and thus perturbed the required
orthogonal orientation of the plane defined by C
, C
, and C
of
the amino acid substrate relative to the plane defined by the
system of the coenzyme-substrate adduct including C
(Scheme 1; Ref.
33). This notion agrees with previous studies by Faleev et
al. (34) who have reported that aspartic and glutamic acid are not
substrates but, in view of the low hydrophobicity of their side chains,
anomalously strong inhibitors of TPL (Ki = 3.5 and
5.0 mM, respectively). We concluded that the introduction
of an arginine residue into position 283 of TPL together with the
substitution of Arg-100 with an uncharged residue, i.e. the
double mutation R100T/V283R, might mimic the binding site for
dicarboxylic substrates of AspAT and thus result in a corresponding
alteration in the substrate specificity of TPL.
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Expression of Wild-type TPL and Mutant TPLs--
The C-terminal
His6 tag did not interfere with the -elimination
activity of the enzyme (Table I). The
His-tagged TPL R100T/V283R enzyme and the single mutant TPL R100T were
purified and used for analysis. The single mutant V283R enzyme,
however, could not be expressed as soluble protein. The PLP content of
the mutant proteins was found to be 1 mol/mol of subunit, as has been
shown previously for wild-type TPL (36). The UV-visible spectrum of the
PLP form of the mutant enzymes is almost identical to that of the
wild-type enzyme. Apparently, the topochemistry of the PLP-binding site
is not significantly altered by the mutations.
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Wild-type TPL in the presence of L-tyrosine exhibits a
visible absorbance peak at about 500 nm attributable to the quinonoid coenzyme-substrate adduct 2 (Scheme 1; Ref. 8). Some other
amino acids such as L- and D-alanine,
L-phenylalanine, L-aspartic acid,
L-methionine, and L-homoserine, which are not
substrates for -elimination, form stable quinonoid intermediates
with wild-type TPL (34). TPL R100T also produced stable quinonoid
intermediates upon addition of these amino acids. However, with TPL
R100T/V283R no detectable quinonoid adduct was observed in the presence
of any amino acid including L-tyrosine.
Changes in Substrate Specificity--
TPL R100T/V283R and TPL
R100T were tested for -elimination activity toward
L-tyrosine and dicarboxylic amino acids of various lengths
(Table I). The kcat value of TPL R100T/V283R
toward L-tyrosine was decreased 30-fold as compared with
wild-type TPL without significant change in the Km
value. When TPL R100T/V283R was tested for activity toward dicarboxylic
amino acids using the coupled assay with lactate dehydrogenase and
NADH, pyruvate was detected in the reaction mixtures. Thin layer
chromatographic analyses confirmed the production of pyruvate. A yellow
spot, the Rf value of which was the same as that of
authentic pyruvate, was detected on the plate as the unique and
invariable oxo acid product of the enzymic reactions with all
dicarboxylic substrates. No 2-oxobutyric acid, which possibly might
have been produced by a
-elimination reaction of
L-glutamate, was detected.
The expected products of the -elimination reaction of the
dicarboxylic substrates L-aspartate,
L-glutamate, and L-2-aminoadipate are pyruvate,
ammonia, and the monocarboxylic acids formate, acetate, and propionate,
respectively. In the case of L-aspartate, formate was
identified and determined using the coupled assay with formate dehydrogenase and NAD+. Equimolar amounts of pyruvate and
formate were detected (Table II). Thus,
TPL R100T/V283R catalyzes, in contrast to the wild-type enzyme, the
-elimination reaction of dicarboxylic substrates at least as
efficiently or, in the case of L-aspartate, even two times
faster than that of L-tyrosine (kcat = 0.21 s
1; Table I). The Km value of
TPL R100T/V283R for the
-elimination reaction with
L-glutamate was significantly lower than the
Km values with L-aspartate and
L-2-aminoadipate. It seems that L-glutamate has
the optimum size for binding among these dicarboxylic substrates. TPL
R100T also catalyzed the
-elimination reaction of the dicarboxylic
amino acids L-aspartate, L-glutamate, and
L-2-aminoadipate; however, the reaction was up to six times slower than that with the double mutant enzyme. The
Km values were also higher (up to 3 times, in the
case of L-glutamate) as compared with the double mutant
enzyme. Furthermore, the TPL R100T-catalyzed
-elimination reaction
of L-tyrosine was five times faster than the TPL
R100T/V283R-catalyzed reaction.
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We also examined the reaction of TPL R100T/V283R and TPL R100T with
3-chloro-L-alanine, L-serine,
S-methyl-L-cysteine, and S-(o-nitrophenyl)-L-cysteine which
are good substrates for -elimination by wild-type TPL (4, 5). The
activity toward 3-chloro-L-alanine was only slightly lower
than that of the wild-type enzyme (Table I). However, no measurable
activity was observed with the other substrates. L-Cysteine
sulfinate, which can be considered an analog of
L-aspartate, was inert as substrate with both mutant TPL enzymes.
Changes in Reaction Specificity-- TPL R100T/V283R was also tested for newly generated catalytic activities with a sensitive assay that monitors both the consumption of the amino acid substrate and the formation of new products (Table III). TPL R100T/V283R in its pyridoxal form underwent the transamination half-reaction with L-aspartate and L-glutamate 10 times faster than the wild-type enzyme. It also transaminated L-serine and L-alanine two times faster than the wild-type enzyme. The racemization of L-alanine catalyzed by TPL R100T/V283R is four times slower than that catalyzed by the wild-type enzyme.
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DISCUSSION |
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X-ray crystallographic analysis has identified Arg-292 as the key
residue in determining the specificity of AspAT for dicarboxylic substrates (12) through direct salt bridge-hydrogen bond interactions with the - or
-carboxylate groups of the substrate. Indeed, replacement of Arg-292 with an aspartate residue has converted E. coli AspAT from an anionic to a very slow cationic amino acid transaminase (37). Similarly, substitution of Arg-292 with valine or
leucine has been found to switch the specificity in favor of aromatic
amino acids (38). Recently, the importance of the guanidinium group of
arginine has also been pointed out by the marked decrease in both the
affinity and the catalytic activity toward dicarboxylic substrates upon
replacement of Arg-292 with lysine (39).
Here, in an attempt to change the substrate specificity of TPL from
L-tyrosine to dicarboxylic substrates, we introduced an arginine residue in TPL in the same position that, as indicated by
homology modeling, is occupied by Arg-292 in AspAT. Measurements of
enzymic activities demonstrated indeed the conversion of TPL, a
tyrosine -lyase, to a dicarboxylic amino acid
-lyase. TPL R100T/V283R catalyzes the
-elimination reaction of
L-aspartate at a 2-fold higher rate than that of
L-tyrosine, the rate of
-elimination of
L-aspartate being only 1 order of magnitude slower than
that of
-elimination of L-tyrosine by the wild-type enzyme.
The dicarboxylic amino acids L-aspartate,
L-glutamate, and L-2-aminoadipate are converted
to pyruvate, ammonia, and the respective monocarboxylic acids,
e.g. with L-aspartate as substrate, production of formate was observed. The pathway of formate production from L-aspartate by -elimination corresponds to that followed
by the wild-type enzyme with its natural substrate
L-tyrosine (Scheme 1). Upon formation of the quinonoid
intermediate 2, the coenzyme donates electrons to the
substrate resulting in the cleavage of the bond between C
and the
nucleophilic leaving group thus producing the aminoacrylate
coenzyme-substrate adduct 3. The cleavage of the aliphatic
C-C bond very likely is facilitated by an active-site group which
protonates the carbanion of the leaving carboxylic acid in a concerted
fashion. Transimination of the aminoacrylate intermediate leads to the
production of ammonium pyruvate 4, restoring the internal
aldimine. The fact that no L-alanine was found in the
reaction mixture together with the production of equimolar amounts of
pyruvate and formate demonstrates that no
-decarboxylation occurred.
Thus,
-elimination and slow transamination of
L-aspartate (Table III) are the only reactions taking
place. Analogous results have been obtained with
L-glutamate and L-2-aminoadipate as substrates.
It has to be noted that the known PLP-dependent
carbon-carbon lyases acting on dicarboxylic amino acids are
decarboxylases, e.g. glutamate decarboxylase (40) and
aspartate
-decarboxylase (41, 42), that produce CO2
and the cognate monocarboxylic amino acid. The reaction observed here reflects a newly generated catalytic activity for
PLP-dependent enzymes toward dicarboxylic amino acids. TPL
R100T/V283R and TPL R100T are the first B6 enzymes that
catalyze the
-elimination reaction of these substrates.
The importance of the introduction of an arginine residue at position
283 for the recognition of dicarboxylic substrates is evident from a
comparison of TPL R100T/V283R with TPL R100T. The single mutant TPL
also catalyzes the -elimination reaction of dicarboxylic substrates;
however, the double mutant TPL R100T/V283R reacts faster with
L-aspartate, and its Km value for L-glutamate is lower (Table I). Moreover, the data agree
with the hypothesis that Arg-100 is the positively charged group in the
active site of TPL that interacts with the distal carboxylate group of
L-aspartate or L-glutamate making them
potential inhibitors but not substrates of wild-type TPL. This mode of
inhibition of TPL by dicarboxylic acids has been proposed previously by
Faleev et al. (34). Replacement of Arg-100 with threonine
renders dicarboxylic substrates more flexible in the active site. The
additional introduction of Arg-283 might stabilize the side chain
carboxylate group in a more favorable position for reaction due to salt
bridge-hydrogen bond interactions similar to those in AspAT (Fig.
4).
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Recent studies (43) have demonstrated that Arg-381 in TPL is required for the recognition of L-tyrosine as substrate. We found that not only the R381I enzyme but also the triple mutant TPL R100T/V283R/R381I has no measurable activity toward both L-tyrosine and dicarboxylic substrates (data not shown).
Apparently, dicarboxylic substrates adopt in the active site of TPL R100T/V283R a similar configuration as L-tyrosine in wild-type TPL and interact in a similar way with the critical residues of TPL that control reaction specificity. Even though the overall catalytic activity of TPL R100T/V283R toward L-tyrosine is decreased, its Km value for this substrate is almost unchanged (Table I) suggesting that the side chain of Arg-283 contributes to the binding of the aromatic ring of L-tyrosine. Such interactions have been reported to apply in E. coli aromatic amino acid aminotransferase (44). How does the side chain of Arg-283 contribute to the recognition of L-tyrosine? The model of the "arginine switching" mechanism (45) assumes that the side chain of arginine moves out of the active site, when aromatic monocarboxylic substrates are bound. This model has been verified by x-ray crystallographic analysis of aspartate aminotransferase that has been engineered into a tyrosine aminotransferase (19, 46). Another model assumes that the guanidinium group of arginine directly links up with the aromatic ring of the bound substrates. This interaction may be energetically favorable (47).
The rate of the transamination half-reaction of dicarboxylic substrates
is increased ten times by the two conjoint mutations. The values of
kcat for the transamination of all the
substrates studied here are in the order of 103
s
1. Apparently, the transamination reactions of these
substrates depend on a rate-limiting step that follows the formation of
the quinonoid intermediate 2. The
kcat values of TPL R100T/V283R for
transamination by and large coincide with those of the wild-type enzyme
(Table III), indicating again that different active-site residues are
important for the catalysis of
-elimination and transamination reactions.
In conclusion, the newly generated substrate specificity of TPL
R100T/V283R agrees with previous studies in which the substrate specificity of B6 enzymes was changed without altering the
reaction specificity by replacement of some critical active-site
residues. The results are thus also consonant with evolutionary studies indicating that the B6 enzymes originated from
regio-specific catalysts, which first specialized for reaction
specificity and then for substrate specificity (2). The results of
previous attempts to change the substrate specificity of aspartate
aminotransferase by both site-directed mutagenesis (19) and forced
molecular evolution (48) suggest that further improvement of the
engineered dicarboxylic amino acid -lyase, i.e.
enhancement of its substrate binding affinity and catalytic efficacy,
would require substitutions of numerous amino acid residues that do not
participate in the active site.
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. R. S. Phillips for useful discussions and for providing us with the plasmid pTZTPL, the expression system, and S-(o-nitrophenyl)-L-cysteine.
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FOOTNOTES |
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* This work was supported in part by Swiss National Science Foundation Grant 31-45940 and the EMDO-Stiftung, Zürich, Switzerland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Biochemisches Institut
der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Tel.: 41-1-6355511; Fax: 41-1-6356805;
E-mail: christen{at}biocfebs.unizh.ch.
The abbreviations used are: TPL, tyrosine phenol-lyase; AspAT, aspartate aminotransferase; PLP, pyridoxal 5'-phosphate; PMP, pyridoxamine 5'-phosphate.
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REFERENCES |
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