Osaka 564-8680, Japan
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ABSTRACT |
The lysine residue binding with the cofactor
pyridoxal 5'-phosphate (PLP) plays an important role in catalysis, such
as in the transaldimination and abstraction of
-hydrogen from a
substrate amino acid in PLP-dependent enzymes. We studied
the role of Lys39 of alanine racemase (EC 5.1.1.1)
from Bacillus stearothermophilus, the PLP-binding residue
of the enzyme, by replacing it site-specifically with alanine and
characterizing the resultant K39A mutant enzyme. The mutant enzyme
turned out to be inherently inactive, but gained an activity as high as
about 0.1% of that of the wild-type enzyme upon addition of 0.2 M methylamine. The amine-assisted activity of the mutant
enzyme depended on the pKa values and molecular volumes of the alkylamines used. A strong kinetic isotope effect was
observed when
-deuterated D-alanine was used as a
substrate in the methylamine-assisted reaction, but little effect was
observed using its antipode. In marked contrast, only
L-enantiomer of alanine showed a solvent isotope effect in
deuterium oxide in the methylamine-assisted reaction. These results
suggest that methylamine serves as a base not only to abstract the
-hydrogen from D-alanine but also to transfer a proton
from water to the
-position of the deprotonated (achiral)
intermediate to form D-alanine. Therefore, the exogenous amine can be regarded as a functional group fully representing Lys39 of the wild-type enzyme. Lys39 of the
wild-type enzyme probably acts as the base catalyst specific to the
D-enantiomer of alanine. Another residue specific to the L-enantiomer in the wild-type enzyme is kept intact in the
K39A mutant.
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INTRODUCTION |
Alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate
(PLP)1 enzyme that occurs
widely in bacteria and plays a central role in the metabolism of
D-alanine, an essential component of the peptidoglycans in
bacterial cell walls. The generally accepted mechanism of alanine
racemase reaction proceeds through the steps shown in Scheme
I. PLP bound with the active-site lysyl
residue (A) reacts with a substrate to form an external
Schiff base (B) through transaldimination. The subsequent
-hydrogen abstraction results in the formation of a resonance-stable
deprotonated intermediate (C). If reprotonation occurs at
the
-carbon of the substrate moiety on the opposite face of the
planar intermediate (C), then an antipodal aldimine
(D) is formed. The
-amino group of the lysine residue is
substituted for the isomerized amino acid through transaldimination,
and the internal aldimine (E) is regenerated. Recent kinetic
analyses (1) and x-ray crystallographic studies (2, 3) have suggested
that the alanine racemase reaction proceeds through a two-base
mechanism; proton donors and proton acceptors are situated on both
sides of the planar intermediate (C) in order to accomplish
removal and return of the
-hydrogen of the substrate amino acid.

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Scheme I.
Proposed mechanism of the alanine racemase
reaction. A, an internal aldimine of PLP with a lysyl
residue; B, an external aldimine of PLP with
D-alanine; C, a quinonoid intermediate formed
after removal of -hydrogen from alanyl external aldimines
B or D; E, an internal aldimine of PLP
with L-alanine.
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The lysyl residue forming a Schiff base with PLP is shown to act as the
base to abstract
-hydrogen from a substrate amino acid in many PLP
enzymes. The lysyl residue binding PLP in alanine racemase is also
believed to play this role (2, 3). However, the PLP-binding lysyl
residue has another important function as a catalyst for
transaldimination with a substrate amino acid. Therefore, one may
expect complicated results by site-directed mutagenesis of the lysyl
residue. However, Toney and Kirsch (4) have developed an elegant method
named chemical rescue by which the function of a lysine residue lost by
mutagenesis can be rationally compensated by means of various kinds of
amines. We have studied the role of lysine 39, the PLP-binding lysyl
residue of alanine racemase from Bacillus stearothermophilus
(BSAR) (5-7), by chemical rescue studies of its K39A mutant (K39A
BSAR) with exogenous amines. We here show that Lys39 of
BSAR probably serves as the base abstracting
-hydrogen specifically from the D-enantiomer of alanine.
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EXPERIMENTAL PROCEDURES |
Materials--
The K39A BSAR was constructed and purified to
homogeneity as described previously (8). The wild-type enzymes of BSAR
(5) and D-amino acid aminotransferase (9) were purified as
described in literature. Alanine dehydrogenase was a gift from Dr. H. Kondo of Unitika Ltd., Osaka, Japan; L-lactate
dehydrogenase was from Boehringer Mannheim.
D-[
-2H]Alanine was prepared from a
reaction mixture (1.0 ml) containing 0.1 M boric acid
buffer (p2H 8.4), 89.1 mg of D-alanine, and 100 units of D-amino acid aminotransferase, whose solvent was
replaced by 2H2O by repeated concentrations and
dilutions with 20 mM potassium phosphate buffer in
2H2O (p2H 8.4) with a Centricon 10 ultrafiltration unit. The reaction was performed at 37 °C for
16 h, and then stopped by heating at 100 °C for 10 min. After
centrifugation, the supernatant solution was applied to a Dowex 50 (formate form) column (inner diameter, 2.0 × 10 cm), and
D-[
-2H]alanine was eluted with 50 ml of
0.1 M HCl. The fractions containing D-alanine
were pooled and evaporated to dryness.
L-[
-2H]Alanine was prepared with
L-methionine
-lyase (10) in a mixture (p2H
7.5 in 1.0 ml of 2H2O) containing 89.1 mg of
L-alanine and 23.7 mg of L-methionine
-lyase. The reaction was carried out at 28 °C for 72 h, and
then L-[
-2H]alanine was isolated in the
same manner as described above.
The deuterium contents of D-[
-2H]alanine
and L-[
-2H]alanine were both higher than
97% when determined by 1H NMR.
Protein Assays--
Protein concentrations were determined by
measurement of absorbance at 280 nm or by the method of Bradford (11)
with bovine serum albumin as a standard. The absorption coefficients at
280 nm were estimated from the molecular weight and the amino acid composition of the enzymes.
Enzyme Assays--
Conversion of D-alanine to
L-alanine catalyzed by BSAR was determined by following the
formation of NADH in a coupled reaction with L-alanine
dehydrogenase. The assay mixture contained 100 mM CAPS
buffer whose pH was adjusted to pH 10.5 with tetramethylammonium hydroxide, 0.15 units of alanine dehydrogenase, 30 mM
D-alanine, and 2.5 mM NAD+ in a
final volume of 1.0 ml. The reaction was started by addition of alanine
racemase (about 0.01 µg) after preincubation of the mixture at
37 °C for 15 min. An increase in absorbance at 340 nm was followed.
One unit of the enzyme was defined as the amount of enzyme that
catalyzed the racemization of 1 µmol of substrate/min.
D-Alanine formed from L-alanine was assayed
with D-amino acid aminotransferase. The assay mixture
contained 100 mM CAPS buffer (adjusted to pH 10.5 with
tetramethylammonium hydroxide), 30 mM L-alanine, 5 mM
-ketoglutarate, 0.16 mM NADH, 12 units of D-amino acid
aminotransferase, and 10 units of lactate dehydrogenase. The reaction
was started by addition of alanine racemase (about 0.01 µg) after
preincubation of the mixture at 37 °C for 10 min. A decrease in
absorbance at 340 nm was monitored.
The amine-assisted reactions were conducted with about 52 µg of K39A
mutant enzyme in the presence of various concentrations (0-200
mM) of amines, whose solutions were adjusted to pH 10.5 with tetramethylammonium hydroxide. The final ionic strength of the reaction mixture was adjusted to 0.5 by addition of
tetramethylammonium chloride.
The apparent rate constant (kobs) of the
reaction is expressed by the following equation (4).
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(Eq. 1)
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kB is the rate constant of the reaction
catalyzed by the deprotonated form of amine;
ksol, the rate constant of the reaction proceeding independently of amine; and [amine]free, the
concentration of deprotonated amine. Since [amine]free
depends on the ionization constant (Ka) of the
amine and proton concentration [H+] in the system,
Equation 1 is converted to Equation 4 as follows.
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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[amine]total is the concentration of added amine,
and [amine]protonated is the concentration of the
protonated form of the amine.
The rate constant, kB, was obtained from the
plots of kobs against
[amine]total/(1 + [H+]/Ka).
Measurement of Isotope Effect--
The substrate deuterium
isotope effect was determined from the rates of racemization of
D- or L-[
-2H]alanine in the
presence of 100 mM methylamine. The conditions were the
same as described above.
The solvent deuterium isotope effect was determined with the assay
mixture (0.5 ml) that contained, in 2H2O,
various concentrations of D- or L-alanine, 100 mM CAPS buffer (p2H 10.9), and 100 mM methylamine. The reaction was started by addition of
alanine racemase, which had been freed from H2O by repeated lyophilization and dissolution in 2H2O, and
stopped by heating at 100 °C for 10 min. L-Alanine
formed was determined as follows. A mixture (0.5 ml) containing 100 mM CHES buffer (pH 9.0), 5 mM NAD+,
0.15 units of alanine dehydrogenase, and an aliquot of the sample solution was incubated at 37 °C for 60 min, and then NADH formed was
measured at 340 nm. D-Alanine produced was assayed in the same manner with a mixture containing 100 mM Tris-HCl
buffer (pH 8.5), 0.2 mM NADH, 5 mM
-ketoglutarate, 0.24 units of D-amino acid
aminotransferase, 2.8 units of lactate dehydrogenase, and an aliquot of
the sample solution. The incubation was done at 37 °C for 60 min.
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RESULTS |
Spectrophotometric Properties of K39A BSAR--
K39A BSAR was
identical with the wild-type BSAR in far-UV (180-200 nm) and near-UV
(200-300 nm) CD spectra (data not shown). This suggests that K39A BSAR
has a secondary structure virtually identical to that of the wild-type
enzyme. K39A BSAR lacks Lys39, which inherently forms a
Schiff base with PLP, but the enzyme showed an absorption maximum
around 410 nm at pH 7.2, which maximum is slightly red-shifted in
comparison with that of a free aldehyde form of PLP. This may have been
due to the formation of a Schiff base between an unknown lysyl residue
and PLP in K39A BSAR, and we examined this possibility by incubating
K39A BSAR with 0.5 M NaBH4 at 25 °C for
8 h. The absorption band at 410 nm disappeared and a new band
appeared at around 330 nm, indicating that the bound PLP was reduced by
the treatment. However, the PLP derivative was removed completely by
dialysis against 6 M guanidine hydrochloride (data not
shown). This indicates that the aldehyde group of PLP stays in a free
form in K39A BSAR. The mutant enzyme had no CD band due to the bound
PLP, in clear contrast to the wild-type enzyme, which showed a negative
CD band around 420 nm (Fig. 1).

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Fig. 1.
Absorption (A) and circular
dichroism (B) spectra of the wild-type and the K39A
BSAR. Absorption spectra of the wild-type (a) and K39A
(b) BSARs were taken in 20 mM potassium
phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at a
protein concentration of 1.0 mg/ml. CD spectra of the wild-type enzyme
(a), and the K39A BSAR in the absence (b) or
presence (c) of 500 mM methylamine were taken in
20 mM CAPS buffer (pH 10.5) containing 0.01%
2-mercaptoethanol at a protein concentration of 2.0 mg/ml with a JASCO
J-600 recording spectropolarimeter at 25 °C with a 0.1- or 1.0-cm
light path length cell under nitrogen atmosphere.
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Effects of Primary Amines on K39A BSAR--
K39A BSAR was
inactive, but its catalytic activities in both directions of
racemization (from L- to D-alanine, and from
D- to L-alanine) were restored by addition of
0.2 M methylamine. In contrast, the wild-type BSAR was
inhibited by methylamine, although only slightly. A negative CD band
similar to that of the wild-type BSAR appeared around 420 nm by
addition of methylamine (Fig. 1). This suggests that methylamine forms
a Schiff base with the C4' of PLP in the same manner as
Lys39 of the wild-type BSAR. Shaw et al. (2)
have reported that the position of PLP-binding lysine in BSAR is very
similar to that in D-amino acid aminotransferase (12) upon
superposition of their bound PLP. The two enzymes are similar, in that
their CD bands at around 420 nm due to bound PLP appear as negative bands. X-ray crystallographic analysis has shown that the lysines in
both enzymes approach the re face of bound PLP (2, 12). Therefore, methylamine forming a Schiff base with PLP in BSAR K39A
presumably approaches the re face of PLP as well.
Brønsted Analysis of the Amine-assisted Racemization Catalyzed by
BSAR K39A--
The activities in both directions of alanine
racemization catalyzed by K39A BSAR increased proportionally with
increases in the concentration of methylamine added (Fig.
2). We found that other alkylamines were
also effective as catalysts (Figs. 3 and 4), although the dialkylamines,
dimethylamine, and diethylamine were ineffective (Fig. 2). However, no
evidence for saturation (at free base concentrations up to 200 mM) was observed, and there appeared to be no significant
binding of alkylamines by K39A BSAR under our experimental conditions.
The rate of the methylamine-assisted reaction at pH 10.5 was much
higher than that at pH 9.0 (Fig. 2). This indicates that only the free
base of alkylamine participates in the catalysis, and we therefore
calculated the rate constants of the reactions due to the free forms of
amines (kB) on the basis of their dissociation
constants (Ka) and pH values of the reaction media, as described under "Experimental Procedures." Toney and Kirsch (4) have shown that a Brønsted analysis is applicable to the
proton transfer catalyzed by K258A mutant aspartate aminotransferase assisted by exogenous amines, and found a multiple linear relationship between log kB and two independent factors, the
pKa and molecular volume of the amine, as
follows.
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(Eq. 5)
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c is the constant term. We also found that a plot of
log kB versus
pKa values of four kinds of amines with similar
molecular volumes exhibited a linearity in both directions of alanine
racemization (Fig. 3). The least squares fit of the data shown in Fig.
3 gave a Brønsted
value of 0.57 (r = 0.85) for the
D
L reaction and of 0.62 (r = 0.92) for the L
D
reaction. Moreover, we found that the log kB
values were inversely proportional to the molecular volumes of a series
of alkylamines with similar pKa values (Fig. 4). The V values calculated from the data shown in Fig.
4 were
0.037 Å3
(r = 0.98) for the D
L reaction and
0.038
Å3 (r = 0.99) for the L
D
reaction.

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Fig. 2.
Amine-assisted racemization catalyzed by the
K39A BSAR. The specific activities of the K39A mutant enzyme for
the reactions from D- to L-alanine
(A), and L- to D-alanine
(B) were measured in the presence of various concentrations
of methylamine at pH 9.0 ( ) and 10.5 ( ), dimethylamine at pH 10.5 ( ), and diethylamine at pH 10.5 ( ).
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Fig. 3.
Relationship between
pKa values of amines with similar
molecular volumes and log kB of
amine-assisted reactions from D- to L-alanine
(A) and L- to D-alanine
(B) catalyzed by K39A BSAR. The
pKa values were taken from the previous
report of Toney and Kirsch (4). The molecular volumes of amines
reported (4) are as follows: ethanolamine, 71.5 Å3;
2-fluoroethylamine, 64.4 Å3; 2-cyanoethylamine, 70.5 Å3; 2,2,2-trifluoroethylamine, 71.6 Å3.
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Fig. 4.
Relationship between molecular volumes of
amines with similar pKa values and log
kB of amine-assisted reactions from
D- to L- alanine (A) and
L- to D-alanine (B) catalyzed
by K39A BSAR. The molecular volumes were taken from the previous
report of Toney and Kirsch (4). The pKa values of
amines reported (4) are as follows: methylamine, 10.6; ethanolamine,
10.6; propylamine, 10.5; butylamine, 10.6.
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Fig. 5.
-Deuterium substrate isotope
effect on amine-assisted reactions catalyzed by K39A BSAR. The
apparent rate constants in the reactions from D to
L-alanine (A) and L- to
D-alanine (B) with non-labeled ( ) or
-deuterated D- or L-alanine ( ) as
substrates were plotted against the concentrations of deprotonated
methylamine.
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Isotope Effect Studies--
Faraci and Walsh (13) have
demonstrated that the step of transaldimination is rate-limiting in the
reaction catalyzed by the wild-type BSAR. Therefore, the rescue effect
by alkylamines observed here may be primarily due to their action as a
base (or a nucleophile) in transaldimination. This is also supported by our findings of good agreement between the two Brønsted
values of
the D
L and the L
D reactions. Faraci and Walsh (13) found that
the isotope effect was virtually absent in the alanine racemization
catalyzed by the wild-type BSAR. However, we examined the deuterium
isotope effect in the methylamine-assisted racemization catalyzed by
K39A BSAR with D- and
L-[
-2H]alanine. As shown in Fig. 5, we
found a significant isotope effect for the reaction from
D-alanine to L-alanine; a clear isotope effect
was visualized on V rather than V/K
(Fig. 5 and Table I). However, no isotope
effect was observed in the L
D reaction. Therefore, the step of
-deuteron abstraction from D-alanine was kinetically
important and probably at least partially rate-limiting throughout the
whole process, while the removal of
-deuteron from the antipode was
kinetically insignificant. In marked contrast, however, when we
examined the solvent isotope effect, a strong effect was observed only
for the reaction from L-alanine to D-alanine (Table I). Thus, the step of
-protonation (with deuterium) to produce D-alanine is kinetically crucial, while its
counterpart for the production of L-alanine from
D-alanine is not. Presumably, the overall profile of the
K39A BSAR reaction assisted by an alkylamine is fairly asymmetric, as
opposed to that of the wild-type BSAR reaction depicted by Faraci and
Walsh (13).
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Table I
Deuterium isotope effects of the methylamine-assisted racemization of
D- and L-alanine catalyzed by the K39A mutant
enzyme
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DISCUSSION |
Toney and Kirsch (4) developed a general approach in which the
activity of a mutant enzyme lacking a catalytically important residue
can be restored by the addition of small molecules functionally equivalent to the missing catalytic group. Since their report, this
"chemical rescue" approach has been successfully applied to a
number of enzymes to identify catalytic residues and to establish structure-activity relationships. The rescuer molecules used are amines
for lysine-mutated enzymes (14-16), guanidines for arginine-mutated enzymes (17, 18), phenol for a tyrosine-mutated enzyme (19), azide or
formate for a glutamate-mutated enzyme (20), and imidazole for
histidine-mutated enzymes (21, 22). In this study, we have demonstrated
by combination of chemical rescue studies and isotope effect analysis
of the amine-assisted reactions catalyzed by K39A BSAR that
Lys39 of the wild-type BSAR probably mediates not only
transaldimination but also
-hydrogen abstraction from the substrate.
This is one of the rare examples of a chemical rescue study clarifying
multiple functions of a catalytically important residue by combination with isotope effect analysis.
The dad B and alr alanine racemases from
Salmonella typhimurium are distinct from BSAR in that the
former enzymes show substrate and solvent deuterium isotope effects
(13). Abstraction of
-deuteron from
D-[
-2H]alanyl-PLP aldimine and protonation
(with deuteron) of the carbanion intermediate (Scheme I) to give the
D-alanyl aldimine in 2H2O are
rate-limiting (although maybe only partially) in the racemization reaction catalyzed by the Salmonella enzymes. Although the
isotope-effect values vary widely among these enzymes, the
amine-assisted reaction catalyzed by K39A BSAR is identical to the
reactions catalyzed by the Salmonella enzymes in this respect.
Sawada et al. (1) have proposed on the basis of kinetic
evidence that the reaction catalyzed by BSAR proceeds through a two-base mechanism. Shaw et al. (2) have postulated on the basis of x-ray crystallographic studies that Lys39 and
Tyr265 of BSAR serve as the bases to abstract
-hydrogen
from the alanyl-PLP aldimine and to protonate the carbanion
intermediate to give the alanyl-PLP aldimine. After
-hydrogen is
abstracted from the alanyl-PLP aldimine by one base, the other base,
which lies on the opposite side of the plane of the alanyl-PLP
carbanion intermediate, probably protonates the carbanion in order for
racemization to occur. Tyr265 is the only possible residue
whose side chain lies almost directly opposite the side chain of
Lys39, and the hydroxyl group of Tyr265 is
postulated to serve as the second base (2). The chemical rescue studies
presented here strongly suggest that Lys39 serves as the
base that abstracts
-hydrogen from the D-alanyl-PLP aldimine and protonates the carbanion intermediate to produce the
PLP-aldimine of the same enantiomer. Therefore, Tyr265 is
assumed to be the second base specifically acting on the
L-alanyl-PLP aldimine. The crystal structure of the BSAR
complex with R-1-aminoethylphosphonic acid
(L-Ala-P) (3), a tight-binding inhibitor of BSAR (23), has
demonstrated that the phenolic oxygen of Tyr265 is located
at a position much closer than the NZ of Lys39 to the
-carbon of the L-Ala-P-PLP aldimine; the phenolic oxygen of Tyr265 is appropriately aligned for proton abstraction
from an L-isomer in the active site of the clarified structure.
Both Lys39 and Tyr265 are conserved in both
dad B and alr alanine racemases of S. typhimurium (24, 25), and these residues probably play the same
roles, respectively, as those in BSAR. According to the kinetic isotope
effect studies of Faraci and Walsh (13), we can assume that
Lys39 is less effective than Tyr265 as the base
in the S. typhimurium enzymes. The only marked difference between BSAR and the S. typhimurium enzymes is that
Glu314 of BSAR is replaced by a methionine in both S. typhimurium enzymes. Crystallographic studies (2) have suggested
that Glu314 of BSAR plays an important role at the active
site. Glu314 of BSAR is hydrogen-bonded with a water
molecule, which is hydrogen-bonded to the O3' of the PLP ring.
Moreover, the side-chain OE1 of Glu314 is also
hydrogen-bonded with NH1 of Arg136, which is believed to be
the binding site for the carboxyl group of the substrate alanine. The
lack of a glutamate at this position in the S. typhimurium
enzymes may be responsible for the apparently higher efficiency of
Lys39 than Tyr265 as a base to abstract
-hydrogen from the substrate alanine. Replacement of
Lys39 with alanine probably perturbed the active site
structure of K39A BSAR, even in the presence of the rescuer
alkylamines, and this may be the reason that the
-hydrogen-abstraction step was rate-limiting (although maybe only
partially) in the amine-assisted reaction.
According to the general mechanism of transaldimination, the imino
nitrogen of an internal Schiff base is considered to be non-protonated
and to remove a proton from the protonated form of the amino group of a
substrate amino acid. Thus, the deprotonated amino group of the amino
acid is allowed to attack the 4'-carbon of the internal Schiff base.
The PLP-binding lysyl residue is released from the geminal diamine, and
the first transaldimination is accomplished. The second
transaldimination proceeds through a nucleophilic attack on the
4'-carbon of the external Schiff base by the free amino group of the
lysyl residue to release a product. The PLP-binding lysyl residue is
known to be crucial as the catalyst in product release, as shown by
site-directed mutagenesis studies on aspartate aminotransferase (4),
tryptophan synthase (26), D-amino acid aminotransferase
(27, 28), and aromatic L-amino acid decarboxylase (29); the
product (or substrate) forms a stable Schiff base with PLP and is not
released readily from the enzyme unless the PLP-binding lysine is
present. On the basis of the mechanism described above, we propose a
mechanism of the amine-assisted reaction catalyzed by K39A BSAR in
which three bases mediate the proton transfer by cooperation with
Lys39 and Tyr265 (Scheme
II). B1 transfers a proton
from the amino nitrogen of the geminal diamine intermediate to the
phenolate of Tyr265; B2 protonates and
deprotonates the alkylamine moiety of the geminal diamine
intermediate; B3 transfers a proton from an alkylamine to
the amino acid moiety released from the geminal diamine intermediate. B1, B2, and B3 are possibly
His166, Arg136, and Asp313,
respectively, according to the x-ray structures of BSAR (2, 3),
although this hypothesis cannot be proven definitively until the x-ray
structure of alanine-bound BSAR is determined.

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Scheme II.
Proposed mechanism of amine-assisted
alanine racemization reactions catalyzed by K39A BSAR.
B1, B2 and B3 are the side chains
of amino acid residues presumably participating in proton donation and
abstraction. The phenolic hydroxyl group of Tyr265 is
assumed to be the second base abstracting -hydrogen from alanyl-PLP
aldimine and donating a proton to the -position of the carbanion
intermediate.
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