From the Laboratory of Microbial Biochemistry,
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611 and
¶ Department of Biotechnology, Faculty of Engineering, Kansai
University, 3-3-35, Yamate-Cho, Suita, Osaka 564, Japan
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ABSTRACT |
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Pyridoxal 5'-phosphate-dependent
amino acid racemases of broad substrate specificity catalyze
transamination as a side reaction. We studied the stereospecificities
for hydrogen abstraction from C-4' of the bound pyridoxamine
5'-phosphate during transamination from pyridoxamine 5'-phosphate to
pyruvate catalyzed by three amino acid racemases of broad substrate
specificity. When the enzymes were incubated with (4'S)- or
(4'R)-[4'-3H]pyridoxamine 5'-phosphate in the
presence of pyruvate, tritium was released into the solvent from both
pyridoxamine 5'-phosphates. Thus, these enzymes abstract a hydrogen
nonstereospecifically from C-4' of the coenzyme in contrast to the
other pyridoxal 5'-phosphate-dependent enzymes so far studied,
which catalyze the stereospecific hydrogen removal. Amino acid racemase
of broad substrate specificity from Pseudomonas putida
produced D- and L-glutamate from
-ketoglutarate through the transamination with
L-ornithine. Because glutamate does not serve as a
substrate for racemization, the enzyme catalyzed the nonstereospecific
overall transamination between L-ornithine and
-ketoglutarate. The cleavage and formation of the C-H bond at C-4'
of the coenzyme and C-2 of the substrate thus occurs
nonstereospecifically on both sides of the plane of the
coenzyme-substrate complex intermediate. Amino acid racemase of broad
substrate specificity is the first example of a pyridoxal enzyme
catalyzing nonstereospecific transamination.
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INTRODUCTION |
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Although enzymatic racemization of amino acid is apparently
simple, consisting of a nonstereospecific rearrangement of the substrate -hydrogen, several different types of amino acid racemases are found in microorganisms. Aspartate racemase (EC 5.1.1.13) (1) and
glutamate racemase (EC 5.1.1.3) (2, 3) are independent of cofactors,
and one or more cysteinyl residues play an important role in the
abstraction of the
-hydrogen from the substrate. Phenylalanine
racemase (EC 5.1.1.11), which is involved in gramicidin S synthesis,
utilizes ATP (4). Alanine racemase (EC 5.1.1.1) in several
microorganisms (5), arginine racemase (EC 5.1.1.9) from
Pseudomonas graveolens (6), and amino acid racemases of
broad substrate specificity (EC 5.1.1.10) of Aeromonas punctata (7), Pseudomonas striata (8), and
Pseudomonas putida (9) all depend on pyridoxal 5'-phosphate
(PLP)1.
Faraci and Walsh (10) proposed a mechanism for alanine racemase that is
probably similar to that of other PLP-dependent amino acid
racemases. The reaction is initiated by transaldimination. In this
step, PLP bound with the active-site lysyl residue through an internal
Schiff base (Scheme IA) reacts
with a substrate to form an external Schiff base (B). The
subsequent -hydrogen abstraction results in the formation of a
resonance-stable anionic intermediate (C). If the
reprotonation occurs at C-2 of the substrate moiety on the opposite
face of the planar intermediate to that where the proton abstraction
occurs, an antipodal aldimine is formed (D). The aldimine
complex is subsequently hydrolyzed to form isomerized amino acid and
regenerates the bound PLP (E).
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The random return of hydrogen to the anionic intermediate is a
characteristic of enzymatic racemization among various pyridoxal enzyme
reactions. In aminotransferase reactions, the abstracted hydrogen is
stereospecifically transferred to C-4' of the cofactor, and a ketimine
intermediate is formed (11). The pyridoxamine 5'-phosphate (PMP) form
of the enzyme and a keto acid are produced by hydrolysis of the
ketimine intermediate. Amino acid racemases are reported to catalyze
the transamination as a side reaction (12). The transamination
catalyzed by amino acid racemases can be attained through a sequence
either A B
F (or G)
H or A
B
C
F (or G)
H
(Scheme I). An equivalent route can be delineated for the antipode: E
D
F (or G)
H or E
D
C
F (or G)
H.
In transamination, mutual hydrogen transfer between the substrate and
C-4' of the cofactor occurs. In all previous studies of transaminations
catalyzed by aminotransferases such as L-aspartate aminotransferase (AspAT) (13), L-alanine aminotransferase
(14), dialkylamino acid aminotransferase (15), pyridoxamine:pyruvate aminotransferase (16), D-amino acid aminotransferase
(D-AAT) (17), and branched chain L-amino acid
aminotransferase (BCAT) (17) as well as other pyridoxal enzymes such as
L-serine hydroxymethyltransferase (18),
L-tryptophan synthase (19), L-glutamate
decarboxylase (20), and L-aspartate -decarboxylase (21),
the hydrogen transfer between substrate and cofactor occurs strictly
stereospecifically on the si or re face of the
plane of the anionic intermediate. However, if the transamination
catalyzed by amino acid racemases proceeds as depicted in Scheme I, the
hydrogen transfer should occur nonstereospecifically on both faces of
the planar intermediate. We were therefore interested in the
stereospecificity of the hydrogen transfer during transamination
catalyzed by amino acid racemases as a side reaction.
We provide here the first evidence that hydrogen removal from C-4' of
PMP occurs randomly on both faces of the substrate-cofactor imine plane
during half transamination catalyzed by the amino acid racemases. We
also show that the enzyme catalyzes nonstereospecific overall
transamination between L-ornithine and -ketoglutarate as
well.
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EXPERIMENTAL PROCEDURES |
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Materials-- Amino acid racemases with low substrate specificity from P. putida (9), P. striata (8), and A. punctata (7) were purified as described previously. BCAT of Escherichia coli K-12 (22) was kindly supplied by Professor H. Kagamiyama and Dr. K. Inoue of Osaka Medical College, Takatsuki, Japan. AspAT from pig heart was obtained from Boehringer Mannheim (Germany), 3H2O (3.7 GBq/g) was from DuPont. The other chemicals were of analytical grade.
Enzyme and Protein Assays-- The activity of the amino acid racemases was determined by measurement of a change in optical rotation at 365 nm with a Perkin-Elmer 241 polarimeter and also by a coupled assay procedure with D- or L-amino acid oxidase (9). Protein concentrations were determined by dye staining with a Bio-Rad protein assay reagent with bovine serum albumin as a standard.
Preparation of Apoenzyme-- Apo-amino acid racemases were prepared as described previously (7). An appropriate amount of enzyme (1-3 mg) was dialyzed against 10 mM potassium phosphate buffer (pH 8.0) containing 30 mM hydroxylamine and 0.1% 2-mercaptoethanol for 24 h and then dialyzed against 10 mM potassium phosphate buffer (pH 8.0) at 4 °C overnight. The apoenzyme formed was determined by measurement of the activity of the enzyme in the presence or absence of 10 mM PLP. Apo-enzymes recovered about 80% of their initial activities on addition of PLP. Apo-AspAT, apo-D-AAT, and apo-BCAT were prepared as described previously (17).
Spectrophotometric Measurements-- Spectrophotometric measurements were made with a Shimadzu UV-visible recording spectrophotometer UV-260 with a 1.0-cm light path at 25 °C.
Preparation of (4'S)- and
(4'R)-[4'3H]PMP--
(4'S)- and
(4'R)-[4'-3H]PMP were prepared by incubation
of the randomly labeled [4'-3H]PMP with apo-BCAT and
apo-AspAT, respectively, as described previously (17). The specific
radioactivities of (4'S)- and (4'R)-[4'3H]PMP prepared were 1.54 × 106 and 1.35 × 106 dpm/µmol,
respectively. Stereospecificities for labeling were confirmed by
measurement of tritium liberation from both PMPs catalyzed by apo-BCAT
and apo-AspAT in the presence of -ketoglutarate as described
previously (17).
Abstraction of C-4' Hydrogen of PMP during
Transamination--
The reaction mixture (100 µl) contained 10 µmol of potassium phosphate buffer (pH 8.0), 5 nmol of sodium
pyruvate, 1 nmol of (4'S)- or
(4'R)-[4'-3H]PMP, and 5 nmol of each apo-amino
acid racemase. The reaction was carried out at 30 °C for 3 h
and terminated by the addition of 100 µl of 1 M HCl. The
mixture was immediately frozen in liquid nitrogen and dried with a
Speed Vac concentrator. The residue was dissolved with 200 µl of
H2O and subjected to a radioactivity assay. The tritium
released from PMP was expressed as volatile radioactivity, which was
estimated by subtraction of the radioactivity finally remaining from
the radioactivity initially added to the reaction mixture. The reaction
mixture (100 µl) with apo-D-AAT and apo-AspAT contained
10 µmol of Tris-HCl buffer (pH 8.0), 10 nmol of sodium
-ketoglutarate, 1.0 nmol of (4'S)- or
(4'R)-[4'-3H]PMP, and 168 µg of apo-AspAT or
182 µg of apo-D-AAT, respectively. The reaction was
carried out at 30 °C for 15 min and terminated by the addition of
100 µl of 1 M HCl. Other conditions were the same as
those of the reactions with amino acid racemases. Radioactivity was
determined with a Packard Tri-Carb scintillation spectrometer with
Clear-solI (Nacalai Tesque, Japan) as a scintillator.
Transamination between L-Ornithine and
-Ketoglutarate Catalyzed by Amino Acid Racemase from P. putida--
The reaction mixture (1 ml) consisted of 10 µmol of
L-ornithine, 10 µmol of
-ketoglutarate, 0.05 µmol of
PLP, 50 µmol of Tricine buffer (pH 8.5), and 20 µg of enzyme. The
reaction was carried out at 37 °C for 2 h and terminated by
boiling. After centrifugation, amino acids in the supernatant solution
were derivatized to diastereomers with Marfey's reagent (23) and
analyzed by HPLC.
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RESULTS |
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Transamination by Amino Acid Racemases--
When the amino acid
racemase of broad substrate specificity from P. putida was
incubated with L-alanine, the absorption at 420 nm derived
from the internal Schiff base decreased, with a concomitant increase in
the absorption at 330 nm (Fig.
1a). This suggests that the
coenzyme form of the enzyme was converted from PLP to PMP during the
racemization. The reversal from PMP to PLP also occurred on the basis
of a spectral shift. The addition of pyruvate to the PMP form of
enzyme, which was prepared by incubation of PMP with the apo-enzyme,
led to a decrease in absorbance at 330 nm and an increase in that at
420 nm (Fig. 1b). Similar spectral shifts were observed in
the reactions of the PMP form of the amino acid racemases from P. striata and A. punctata with pyruvate (data not shown).
The results indicate that these amino acid racemases catalyze the
transamination as a side reaction. Amino acid racemase of P. putida catalyzes the overall transamination between ornithine and
pyruvate. The specific activity for the transamination was 0.26 units/mg (~10.7 min1). The rate of transamination was
lower than that of the racemization of ornithine by a factor of
1.1 × 104. However, the rate of enzymatic
transamination was at least several orders of magnitude higher than
that of the nonenzymatic transamination, which was lower than the
minimum value for accurate determination, ~4.0 × 10
6 min
1.
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Stereospecificity of Amino Acid Racemases for Hydrogen Abstraction
from C-4' of PMP during Half-transamination--
When the PMP form of
an enzyme is converted to the PLP form by transamination with an amino
acceptor (keto acid), one of the two hydrogens at C-4' of PMP is
usually transferred stereospecifically to C of the keto acid (24).
We studied the stereospecificity of amino acid racemases for hydrogen
abstraction from C-4' of PMP using the method described previously
(17). Stereospecificity is determined by measurement of the
radioactivity of 3H released from the PMPs, which are
stereospecifically tritiated at C-4'. Each 5 nmol of apo-amino acid
racemase was incubated with 1 nmol of (4'S)- or
(4'R)-[4'-3H]PMP and 5 nmol of sodium
pyruvate. We deduce that PMP was completely converted to PLP, because
the PMP form of the amino acid racemase from P. putida
recovered 100% of the activity theoretically expected. As shown in
Table I, tritium was released equally
from both (4'S)- and
(4'R)-[4'-3H]PMPs in the presence of amino
acid racemases. The amount of tritium released from each PMP was about
50% that which initially existed. The control experiment was done with
D-AAT and AspAT. As reported previously (17),
D-AAT and AspAT catalyzed the stereospecific removal of
tritium from (4'R)- or
(4'S)-[4'3H]PMP, respectively, as shown in
Table I. These results confirm the stereospecific tritium labeling of
both PMPs. No tritium was released from each PMP in the absence of
enzyme. Thus, the amino acid racemases catalyze the non-stereospecific
abstraction of hydrogen from C-4' of PMP. They are the first class of
pyridoxal enzyme catalyzing the hydrogen removal on both sides of the
plane of a substrate-cofactor complex during transamination.
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Overall Transamination by Amino Acid Racemase from P. putida--
If the hydrogen is introduced nonspecifically to C-2 of
the keto acid moiety of the anionic intermediate on both sides of the
planar intermediate during the half reaction of transamination, racemic
amino acid is formed from the keto acid (Scheme I; H F (or G)
C
B
A or H
F (or G)
C
D
E). We studied the
stereochemistry of glutamate formed from
-ketoglutarate by transamination with L-ornithine catalyzed by the amino acid
racemase of P. putida. After the reaction, the products were
derivatized to diastereomers with Marfey's reagent (23) and subjected
to HPLC. As shown in Fig. 2, both
enantiomers of glutamate and ornithine were found. The amino acid
racemase from P. putida catalyzes the racemization of
ornithine, but glutamate is inert as a substrate for the racemase
reaction (9). Thus, both enantiomers of glutamate were directly formed
by transamination, not by racemization of one enantiomer produced
through transamination. Amino acid racemase from P. putida
catalyzes the nonstereospecific overall transamination.
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DISCUSSION |
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We here show that nonstereospecific hydrogen abstraction from C-4'
of PMP occurs in the transamination between PMP and pyruvate catalyzed
by amino acid racemases with broad substrate specificity. The enzyme
from P. putida also catalyzes nonstereospecific overall transamination between ornithine and -ketoglutarate. These results indicate that the cleavage and formation of the C-H bonds at C-4' of
the coenzyme occur on both sides of the plane of the anionic intermediate. This model is compatible with the proposed mechanism for
racemization. The removal and return of
-hydrogen of substrate occur
on both sides of the plane (Scheme I). It may be interesting to examine
whether the removal of substrate
-hydrogen and the introduction of
coenzyme C-4' hydrogen occur on the same side of the plane. The
examination will be facilitated by analysis of the configuration of
3H-labeled PMP formed in 3H2O by
transamination with either D or L enantiomer of
an appropriate substrate. However, the amino acid used for this purpose
needs to be inert as a substrate for racemization; otherwise, PMP will be also derived from the antipode of the substrate enantiomer added
initially. Glutamate might be the only candidate substrate for this
purpose; however, this is not the case. Glutamate is inert as an amino
donor in transamination,2
although
-ketoglutarate serves as an amino acceptor as described above.
Two mechanisms have been proposed for enzymatic racemization: a
two-base mechanism and a one-base mechanism (25, 26). In the two-base
mechanism, two different bases participate in the catalysis; one
abstracts the hydrogen from a substrate, and the other returns a
hydrogen to the deprotonated intermediate. Glutamate racemase (27, 28),
aspartate racemase (1), proline racemase (25, 30, 31), and
diaminopimelate epimerase (32), which do not require cofactors,
catalyze racemization by this mechanism. In the one-base mechanism, a
single amino acid residue abstracts the -hydrogen from a substrate
and nonstereospecifically returns it to the anionic intermediate.
A swinging door motion has been proposed by Henderson and Johnston (33)
as a model to fit the one-base mechanism; the plane of the
substrate·PLP complex acts like a swinging door in order that the
base can be located on both faces of the plane. If only a single base
is involved, one can expect that the -hydrogen derived from the
substrate will be retained at the
-position of the product (25).
Such an internal retention of the
-hydrogen was verified in the
reactions catalyzed by two PLP-dependent racemases: amino
acid racemase with low substrate specificity from P. striata (34) and
-amino-
-caprolactam racemase from Achromobacter
obae (35). Thus, it was supposed that these reactions proceed
through a single-base mechanism (34, 35). However, Shostak and Schirch (36) argued in their studies on the mechanism of alanine racemization catalyzed by serine hydroxymethyltransferase that the internal retention of the substrate-derived
-hydrogen could also allow a
two-base mechanism by assuming a hydrogen shuttle between the bases as
proposed for the aconitase reaction; the latter enzyme is known to have
a network of at least five interchangeable protons at the active site
that only exchange slowly with the solvent (37). Although the reaction
catalyzed by the amino acid racemase from P. striata was
accompanied by clear internal retention of the
-hydrogen, a two-base
mechanism has been proposed on the basis of the complete disagreement
between the substrate enantiomers examined for the relative rates of
deuterium incorporation from 2H2O into separate
enantiomers (38). Our findings shown here indicate that the catalytic
base(s) responsible for
-hydrogen abstraction and addition are
situated on both faces of the plane of the substrate-cofactor complex.
Our enzyme is assumed to be closely homologous to the P. striata enzyme, because the latter strain is now classified into
the same group as P. putida. If this is the case, our enzyme
would also use two bases for catalysis. Recently, Shaw et
al. (39) clarified the three-dimensional structure of alanine
racemase from Bacillus stearothermophilus. They suggested that Tyr-265 and Lys-39, the PLP binding lysine, serve as the bases.
Sawada et al. have presented kinetic evidence to show that the alanine racemase reaction follows a two-base mechanism (40).
The stereospecificity for the hydrogen transfer reflects the structure
of the active site of pyridoxal enzymes, especially the topographical
relationship between the catalytic base for the hydrogen transfer and
the bound coenzyme. Therefore, stereospecificity has been discussed in
relation to the molecular evolution of the pyridoxal enzymes (17, 18,
24). No whole primary structure or three-dimensional structure of amino
acid racemases with low substrate specificity has been determined.
However, the primary structures of alanine racemases, which are
homologous to the amino acid racemases in the sequences around the
lysine residue binding PLP (41), show few similarities to those of
other PLP enzymes (42). Therefore, amino acid racemase with low
substrate specificity probably belongs to the same family of proteins
as alanine racemase: a unique family containing only alanine racemase,
mammalian ornithine decarboxylase, and
meso-,
-diaminopimelate (DAP) decarboxylase with
little similarity to other PLP enzymes (43). We have shown that the
decarboxylation of DAP catalyzed by DAP decarboxylase proceeds through
inversion of the configuration of the
-carbon of DAP (29). This
indicates that the enzyme function must be conducted on both sides of
the plane of the substrate·PLP complex so as to decarboxylate on one
side and to introduce a proton on the other side. The DAP decarboxylase
reaction is homologous to the racemase reaction in this respect. It may
be interesting to examine whether DAP decarboxylase and ornithine
decarboxylase catalyze the removal of tritium nonstereospecifically
from C-4' of both (4'S)- and
(4'R)-[4'-3H]PMP in the same manner as amino
acid racemase with low substrate specificity. Our preliminary results
have shown that alanine racemase from B. stearothermophilus
also catalyzes the nonstereospecific removal of tritium from both
[4'-3H]PMPs. Whatever the results of the DAP
decarboxylase and ornithine decarboxylase reactions may be, it is
interesting to note the clear relationship between the
stereospecificity of hydrogen transfer and families of PLP enzymes.
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FOOTNOTES |
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* This work was supported in part by the Research for the Future Program from the Japan Society for the Promotion of Science.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. Tel.: 81-774-38-3240; Fax: 81-774-38-3248; E-mail: esaki{at}scl.kyoto-u.ac.jp.
1
The abbreviations used are: PLP, pyridoxal
5'-phosphate; AspAT, aspartate aminotransferase; BCAT, branched chain
L-amino acid aminotransferase; D-AAT,
D-amino acid aminotransferase; PMP, pyridoxamine 5'-phosphate; Tricine, N-tris(hydroxymethyl)methylglycine;
HPLC, high performance; DAP,
meso-,
-diaminopimelate.
2 Lim, Y.-H., Yoshimura, T., Kurokawa, Y., Esaki, N., and Soda, K, unpublished results.
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REFERENCES |
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