Role of Lysine 39 of Alanine Racemase from Bacillus stearothermophilus That Binds Pyridoxal 5'-Phosphate
CHEMICAL RESCUE STUDIES OF Lys39 right-arrow Ala MUTANT*

Akira WatababeDagger §, Yoichi KurokawaDagger §, Tohru YoshimuraDagger , Tatsuo KuriharaDagger , Kenji Soda, and Nobuyoshi EsakiDagger parallel

From the Dagger  Laboratory of Biofunctional Molecules, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 and the  Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35, Yamate-Cho, Suita, Osaka 564-8680, Japan

    ABSTRACT
Top
Abstract
Introduction
References

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 alpha -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 alpha -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 alpha -hydrogen from D-alanine but also to transfer a proton from water to the alpha -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.

    INTRODUCTION
Top
Abstract
Introduction
References

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 alpha -hydrogen abstraction results in the formation of a resonance-stable deprotonated intermediate (C). If reprotonation occurs at the alpha -carbon of the substrate moiety on the opposite face of the planar intermediate (C), then an antipodal aldimine (D) is formed. The epsilon -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 alpha -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 alpha -hydrogen from alanyl external aldimines B or D; E, an internal aldimine of PLP with L-alanine.

The lysyl residue forming a Schiff base with PLP is shown to act as the base to abstract alpha -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 alpha -hydrogen specifically from the D-enantiomer of alanine.

    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-[alpha -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-[alpha -2H]alanine was eluted with 50 ml of 0.1 M HCl. The fractions containing D-alanine were pooled and evaporated to dryness.

L-[alpha -2H]Alanine was prepared with L-methionine gamma -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 gamma -lyase. The reaction was carried out at 28 °C for 72 h, and then L-[alpha -2H]alanine was isolated in the same manner as described above.

The deuterium contents of D-[alpha -2H]alanine and L-[alpha -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 alpha -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).
k<SUB><UP>obs</UP></SUB>=k<SUB><UP>B</UP></SUB> [<UP>amine</UP>]<SUB><UP>free</UP></SUB>+k<SUB><UP>sol</UP></SUB> (Eq. 1)
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.
[<UP>amine</UP>]<SUB><UP>protonated</UP></SUB>=[<UP>amine</UP>]<SUB><UP>free</UP></SUB> · [<UP>H</UP><SUP>+</SUP>]/K<SUB><UP>a</UP></SUB> (Eq. 2)
[<UP>amine</UP>]<SUB><UP>total</UP></SUB>=[<UP>amine</UP>]<SUB><UP>free</UP></SUB>+[<UP>amine</UP>]<SUB><UP>protonated</UP></SUB> (Eq. 3)
=[<UP>amine</UP>]<SUB><UP>free</UP></SUB>(1+[<UP>H</UP><SUP>+</SUP>]/K<SUB><UP>a</UP></SUB>)
k<SUB><UP>obs</UP></SUB>=k<SUB><UP>B</UP></SUB> [<UP>amine</UP>]<SUB><UP>total</UP></SUB>/(1+[<UP>H</UP><SUP>+</SUP>]/K<SUB><UP>a</UP></SUB>)+k<SUB><UP>sol</UP></SUB> (Eq. 4)
[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-[alpha -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 alpha -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.

    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.

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.
<UP>log</UP> k<SUB><UP>B</UP></SUB>=&bgr;(<UP>p</UP>K<SUB><UP>a</UP></SUB>)+V (<UP>molecular volume</UP>)+c (Eq. 5)
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 beta  value of 0.57 (r = 0.85) for the D right-arrow L reaction and of 0.62 (r = 0.92) for the L right-arrow 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 right-arrow L reaction and -0.038 Å3 (r = 0.99) for the L right-arrow 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 (bullet ) and 10.5 (open circle ), dimethylamine at pH 10.5 (black-square), and diethylamine at pH 10.5 (triangle ).


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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.   alpha -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 (open circle ) or alpha -deuterated D- or L-alanine (bullet ) as substrates were plotted against the concentrations of deprotonated methylamine.

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 beta  values of the D right-arrow L and the L right-arrow 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-[alpha -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 right-arrow D reaction. Therefore, the step of alpha -deuteron abstraction from D-alanine was kinetically important and probably at least partially rate-limiting throughout the whole process, while the removal of alpha -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 alpha -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


    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 alpha -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 alpha -deuteron from D-[alpha -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 alpha -hydrogen from the alanyl-PLP aldimine and to protonate the carbanion intermediate to give the alanyl-PLP aldimine. After alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -hydrogen from alanyl-PLP aldimine and donating a proton to the alpha -position of the carbanion intermediate.

All monoalkylamines (and their derivatives) examined here showed significant rescue effects to enhance the activity of K39A BSAR, and their efficiencies depended on both molecular volume and pKa. However, dialkylamines were ineffective, and in this respect our results differ markedly from those of Kirsch and Toney (30); that is, we found that dimethylamine was also an effective rescuer for the K258A mutant of aspartate aminotransferase. This difference was probably due to a difference in the catalytic role of rescuer amines in the enzymes examined. Amines primarily serve as the base that abstracts alpha -hydrogen from substrate amino acid in aspartate aminotransferase. In K39A BSAR, however, transaldimination is kinetically more important than alpha -hydrogen abstraction as a role of amines; transaldimination is believed to be rate-determining in the wild-type BSAR reaction (13). Dialkylamines are probably less efficient than monoalkylamines as a catalyst of transaldimination for reason of steric hindrance, since amines need to bind covalently to C4' of PLP in transaldimination. However, no covalent bond formation is required in order for amines to abstract alpha -hydrogen from substrate. The Brønsted beta  values observed for both the D right-arrow L and L right-arrow D reactions probably reflect the transition state of either of the two steps in transaldimination: either deprotonation of the protonated form of amino acid substrate by alkylamine to form PLP-aldimine or nucleophilic addition of alkylamine at C4' of the substrate-PLP aldimine. Both of these steps depend on the pKa values of the amines used (31), and we currently have no explanation for the Brønsted beta  values we obtained. Nonetheless, this constitutes the first report of a chemical rescue for a lysyl residue responsible for transaldimination.

    ACKNOWLEDGEMENTS

We are grateful to Professors Hiroyuki Kagamiyama and Hideyuki Hayashi of Osaka Medical College, Professor Ken Hirotsu of Osaka City University, and Professor Seiki Kuramitsu of Osaka University, for their helpful discussions.

    FOOTNOTES

* This work was supported in part by Grant-in-aid for Scientific Research 08680683 (to T. Y.) from the Ministry of Education, Science, Sports and Culture of Japan, and by a research grant from the Japan Society for the Promotion of Science (Research for the Future) (to N. E.).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.

§ These authors contributed equally and should both be considered as first author.

parallel 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.

The abbreviations used are: PLP, pyridoxal 5'-phosphate; BSAR, alanine racemase from Bacillus stearothermophilus; K39A BSAR, BSAR in which the residue Lys39 was replaced by an alanine residue; CD, circular dichroism; CAPS, 3-(cyclohexylamino)propionesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid.
    REFERENCES
Top
Abstract
Introduction
References

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