Active-site Arg right-arrow  Lys Substitutions Alter Reaction and Substrate Specificity of Aspartate Aminotransferase*

(Received for publication, June 9, 1997, and in revised form, June 27, 1997)

Rosa Anna Vacca Dagger , Sergio Giannattasio Dagger , Rachel Graber §, Erika Sandmeier §, Ersilia Marra Dagger and Philipp Christen §

From the Dagger  Centro di Studio sui Mitocondri e Metabolismo Energetico, Consiglio Nazionale delle Ricerche, Bari and Trani, Italy and § Biochemisches Institut der Universität Zürich, CH-8057 Zürich, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Arg386 and Arg292 of aspartate aminotransferase bind the alpha  and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 °C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta -decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg right-arrow Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.


INTRODUCTION

The pyridoxal 5'-phosphate (PLP)1-dependent enzymes that catalyze transformations of amino acids (for a recent review, see Ref. 1) constitute a few families of evolutionarily related enzymes (2). The member enzymes of such a family use the same protein scaffold to catalyze quite diverse reactions. Apparently, subtle structural differences underlie their catalytic specificity.

Aspartate aminotransferase (AspAT) is probably the most extensively studied PLP-containing enzyme. It catalyzes the reversible transamination of the dicarboxylic L-amino acids, aspartate and glutamate, and the corresponding 2-oxo acids, oxalacetate and 2-oxoglutarate. During the catalytic cycle, the cofactor shuttles between the PLP and the pyridoxamine 5'-phosphate (PMP) forms. High resolution x-ray crystallographic analyses (3-6) in conjunction with site-directed mutagenesis studies (7-14) have elucidated the role of several active-site residues. The specificity for dicarboxylic amino acids appears to be based mainly on two active-site arginine residues (Fig. 1). Arg386 of the small domain binds the alpha -carboxylate group of the substrate, and Arg292 of the large domain of the adjacent subunit interacts with the distal carboxylate group. The spatial orientation of these key residues is determined by steric constraints and polar interactions. The van der Waals contacts of the guanidino nitrogens of Arg386 with the side chain carbonyl of Asn194 and the aromatic ring of Phe360 effectively delimit the conformational space available to Arg386. The guanidino nitrogens of Arg292 are within hydrogen bonding distance from the carboxylate group of Asp15, the side chain amide of Asn142, and the hydroxy group of Ser296 of the adjacent subunit. The side chain of Arg292 is thus maintained in an extended configuration, which favors interaction with the distal carboxylate group of the incoming substrate (15, 12). Arg386 is invariant in all known aminotransferase sequences. Arg292 is conserved in most AspAT sequences, and other aminotransferases have variable residues at position 292 (16).


Fig. 1. Stereoview of the active site of Escherichia coli AspAT (external aldimine with 2-methyl-L-aspartate; Ref. 6). The coenzyme-substrate adduct, Arg292 (R292), and Arg386 (R386), which bind dicarboxylic substrates and were substituted by lysine residues in this study; coenzyme-binding Lys258 (K258); and some other relevant residues are shown. The numbering of residues follows that used for the pig cytosolic isoenzyme.
[View Larger Version of this Image (11K GIF file)]

In an attempt to explore the mechanisms responsible for the reaction specificity of AspAT, we re-engineered the substrate-binding site of the enzyme by substituting the substrate-binding Arg292 and Arg386 with lysine residues. This conservative substitution was expected not to abolish the catalytic apparatus of the enzyme, but to alter the electron repartition and certain bond angles in the coenzyme-substrate adduct, both important determinants of catalytic specificity.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis and Purification of Wild-type and Mutant AspATs

Oligonucleotide-directed mutagenesis of the wild-type aspC gene of Escherichia coli inserted into the BS M13 vector (17) was performed with the mutagenesis kit of Bio-Rad (18) and the oligonucleotides GC CAC ATT TAC TTT ACC AGA AGC and GA GTA GTT AGC TTT AAT CGC CGC T for the R386K and the R292K mutation, respectively. The mutations were confirmed by determination of the nucleotide sequences. The mutated DNAs were expressed in the AspAT-deficient E. coli strain TY103 (19) with the expression vector pKDHE19 (20).

Wild-type and mutant enzymes were purified as described previously (13). The enzymes were reconstituted by incubation with 0.2 mM PLP and 2 mM 2-oxoglutarate for 20 min at room temperature in the dark and subsequent gel filtration (Sephadex G-25) in 50 mM 4-methylmorpholine, pH 7.5. The purified enzymes were stored at -20 °C in the same buffer at a concentration of 50-100 mg/ml. Enzyme concentrations were determined on the basis of the absorption coefficient of the subunit epsilon 280 = 4.7 × 104 M-1 cm-1 (21).

Measurement of Aminotransferase Activity

The activity of the purified enzymes toward various amino and oxo acid substrates was determined under single turnover conditions by monitoring the changes in the UV/VIS absorption spectrum of the enzyme-bound cofactor. The reactions were conducted at 25 °C in 50 mM 4-methylmorpholine, pH 7.5, containing 9 µM (subunit concentration) AspAT and the substrate. The half-reaction from amino acid to oxo acid was followed by measuring the decrease in absorbance at 360 nm and the increase in absorbance at 330 nm due to the conversion of enzyme-bound PLP to PMP; the reverse half-reaction was followed in an analogous manner. The PMP form of the enzymes was prepared by incubation of the PLP form with 1 mM PMP and 5 mM cysteine sulfinate for 30 min at 25 °C in the dark followed by Sephadex G-25 chromatography. The reactions were followed with a Beckman 7400 DU spectrophotometer. With rapidly reacting substrates, a stopped-flow apparatus (a pbp-Spectra Kinetic Monochromator 05-109 from Applied Photophysics) with a cuvette of 1-cm path length and a dead time of 2 ms was used. In all cases, the reaction progress curves fitted to single exponential equations with the pseudo-first order rate constant kobs. The values for kcat and Km were obtained from the kobs values at varying substrate concentrations by Lineweaver-Burk analysis using GRAFIT software (Erithacus Software). The calculated values of kcat/Km were in good agreement with the values measured at low substrate concentrations ([S0] <<  Km), where kobs/[S0] gives directly kcat/Km.

Steady-state aspartate aminotransferase activity was measured in a coupled assay with malate dehydrogenase in 50 mM 4-methylmorpholine, pH 7.5, at 25 °C in the presence of 20 mM 2-oxoglutarate plus 20 mM or 150 mM L-aspartate for wild-type enzyme or mutant AspATs, respectively.

Determination of Dissociation Equilibrium Constants for Competitive Inhibitors and of pK'a of Internal Aldimine by Spectral Titration

Spectral titrations of the PLP form of the wild-type and mutant enzymes were performed at 25 °C in 20 mM sodium phosphate. The dissociation equilibrium constants of the wild-type and mutant enzymes for competitive inhibitors were determined at pH 7.5 (9 µM subunit concentration) by monitoring the absorbance of enzyme-bound PLP at 430 nm as a function of inhibitor concentration (22). To determine the pK'a value of the internal aldimine, 3 ml of enzyme solution were titrated by the repeated addition of 3-5 µl of 2 M acetic acid. The ensuing pH values were measured, and spectra were recorded from pH 7.5 to 5.0. In the titration of the wild-type and the three mutant enzymes, the absorption maximum shifted from 360 to 430 nm. The spectral change reflects the protonation of the internal aldimine. The values of Kd for competitive inhibitors and pK'a of the internal aldimine were obtained by fitting the measured values of absorbance at 430 nm to theoretical dissociation curves using GRAFIT software.

Assay for Newly Generated Activities toward Amino Acids

Mutant AspATs and wild-type enzyme (0.9 mM subunit concentration) were incubated in 50 mM 4-methylmorpholine, pH 7.5, at 25 °C with different amino acids and the cognate oxo acids as substrates. Samples of 20 µl were withdrawn at different times and immediately frozen in liquid nitrogen. For quantitative analysis of the reaction products, the samples were deproteinized with 1.12 M perchloric acid, derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (from Pierce; Ref. 23), and loaded onto a reverse-phase high pressure liquid chromatography column (Aquapore RP-300; 250 × 4.6 mm). The separated derivatives of amino acid substrates and products were photometrically detected at 340 nm (24). In the assay for serine dehydratation, samples of the reaction mixture were deproteinized and directly analyzed for pyruvate with lactate dehydrogenase and NADH.


RESULTS

Spectroscopic Properties of Mutant AspATs

Virtually identical absorption and CD spectra in the visible region for wild-type and mutant enzymes, in both their PLP and PMP forms (not shown), indicate that the mutations leave the active-site geometry essentially undisturbed. Spectrophotometric pH titration gave a pK'a value of 6.4 for the internal aldimine of all three mutant enzymes and of 6.3 for the wild-type enzyme. Apparently, the positive electrostatic potential due to Arg292 and Arg386 that is assumed to account for the low pK'a of the internal aldimine (4) is maintained in the Arg right-arrow Lys mutant AspATs.

Changes in Reaction Specificity

Replacement of either Arg386 or Arg292 with a lysine residue resulted in a marked decrease in aminotransferase activity toward dicarboxylic substrates. The kcat values of the half-reaction from L-aspartate to oxalacetate were decreased by 2 orders of magnitude in the single mutant enzymes and by 4 orders of magnitude in the double mutant enzyme (Table I). Due to a general increase in Km values by 1-2 orders of magnitude, an even larger decrease in the catalytic efficiency kcat/Km was observed for all mutant enzymes. The k'cat values of the overall steady-state transamination reactions catalyzed by the mutant enzymes were decreased commensurately with the decrease in rate of the half-reactions (Table II).

Table I. Kinetic parameters for transamination half-reactions of wild-type and mutant AspATs with dicarboxylic, monocarboxylic, and aromatic amino acids

Values were determined under single turnover conditions in 50 mM 4-methylmorpholine, pH 7.5, at 25 °C and 9 µM subunit concentration by Lineweaver-Burk analysis of the rate of the decrease in A360 (see "Experimental Procedures"). The concentration ranges of the tested amino acids were 10-200 mM for dicarboxylic amino acids, 2.5-150 mM for L-alanine, 5-90 mM for L-norleucine, 1-30 mM for L-phenylalanine, and 1-5 mM for L-tyrosine.

Substrates AspAT wild type
AspAT R292K
AspAT R386K
AspAT R292K/R386K
kcat Km kcat/Km kcat Km kcat/Km kcat Km kcat/Km kcat Km kcat/Km

s-1 mM M-1 s-1 s-1 mM M-1 s-1 s-1 mM M-1 s-1 s-1 mM M-1 s-1
L-Aspartate 530a 4a 1.3 × 105a 4.5b 326 14 9.6b 72 133 0.055 300 0.16
L-Glutamate 670a 37a 1.8 × 104a 0.44b 177 2.5 1.4b 300 4.6 0.003 293 0.01
L-2-Aminoadipate 3.4b 118 28 0.16b 126 1.3 0.003 222 0.013 0.001 100 0.01
L-Alanine 0.07 138 0.51 0.005 129 0.04 0.002 152 0.013 0.005 490 0.01
L-Norleucine 0.06 89 0.67 0.14b 12 11.7 0.002 98 0.020 0.003 304 0.01
L-Phenylalanine c c 150b c c 83b c c 0.06 c c 0.04
L-Tyrosine c c 420b c c 250b c c 16 c c 7

a Ref. 7.
b Determined by stopped-flow technique.
c Saturation was not apparent within the concentration range tested. kcat/Km values were obtained by linear least squares regression of the data.

Table II. Increased rates of side reactions of mutant AspATs

The activities of the enzymes (0.9 mM subunit concentration) toward 20 mM L-aspartate, 8 mM oxalacetate; 20 mM L-glutamate, 20 mM 2-oxoglutarate; 40 mM L-alanine, 10 mM pyruvate; and 20 mM L-serine, 10 mM 2- oxoglutarate were measured in 50 mM 4-methylmorpholine, pH 7.5, at 25 °C. For details, see "Experimental Procedures." No reactions of the substrates were observed in nonenzymic controls with 0.9 mM PLP.

Substrates Activities k'cat
AspAT wild type AspAT R292K AspAT R386K AspAT R292K/R386K

s-1
L-Aspartate Transaminationa 220 0.5 2.0 0.03
 beta -Decarboxylation 1.3  × 10-4 20  × 10-4b 4  × 10-4 4  × 10-4
Racemization 3  × 10-5 1  × 10-4 3.4  × 10-5 5  × 10-5
L-Glutamate Racemization 1.5  × 10-5 8  × 10-5 1.2  × 10-5 1.5  × 10-5
L-Alanine Racemization 1.5  × 10-5 3  × 10-5 3  × 10-5 5  × 10-5
L-Serine Dehydratation 2  × 10-5 1.5  × 10-5 3  × 10-5 1.6  × 10-5

a Steady-state assays of overall reaction of transamination (see "Experimental Procedures").
b The increased k'cat value of AspAT R292K corresponds to the rate of the steady-state reaction. With some preparations of this mutant enzyme, faster beta -decarboxylation was observed in the first minutes of the reaction.

The pH rate profiles for AspAT R386K and AspAT R292K/R386K did not significantly differ from that of wild-type enzyme (Fig. 2). In contrast, the pH optimum of AspAT R292K was considerably narrower than that of the wild-type enzyme. The decrease in activity at higher pH might be due to the deprotonation of the newly introduced Lys292. The affinity of AspAT R292K for aspartate decreased only little with pH (Km of Asp = 14 mM at pH 7.5, 18 mM at pH 9.0, and 36 mM at pH 10.0), indicating that kcat rather than Km is affected at higher pH.


Fig. 2. pH rate profile of wild-type and mutant AspATs. Activities were measured with the malate dehydrogenase-coupled assay at 25 °C with 20 mM 2-oxoglutarate plus 20 mM L-aspartate or 150 mM L-aspartate for wild-type and mutant AspATs, respectively. The following buffers were used at 50 mM concentration: Mes, pH 5.5-7.0; Hepes, pH 7.0-8.5; Ches, pH 8.5-10. The differences in activity at the overlaps of two buffers were less than 10% of the total activity; the mean values are indicated. bullet , wild-type AspAT; black-down-triangle , AspAT R386K; black-triangle, AspAT R292K; black-square, AspAT R292K/R386K.
[View Larger Version of this Image (18K GIF file)]

All three mutant enzymes were analyzed for newly generated catalytic activities (Table II). AspAT R292K racemizes L-aspartate, L-glutamate, and L-alanine 3, 5, and 2 times faster, respectively, than the wild-type enzyme. The same mutant enzyme catalyzes the beta -decarboxylation of L-aspartate to L-alanine with a k'cat of 0.002 s-1, i.e. 15 times faster than the wild-type enzyme. The ratio between the steady-state rates of beta -decarboxylation and transamination is 0.004 and 5.9 × 10-7 for AspAT R292K and the wild-type enzyme, respectively. Both AspAT R386K and R292K/R386K exhibited a three times higher beta -decarboxylase activity and a 2-3-fold higher alanine racemase activity than the wild-type enzyme. All three mutant enzymes showed about the same serine dehydratase activity as the wild-type enzyme.

Changes in Substrate Specificity

Aliphatic dicarboxylic and monocarboxylic amino and oxo acids of various length as well as aromatic substrates were tested for transamination (Tables I and III). As previously shown for AspAT R386K (7) and now for AspAT R292K and AspAT R292K/R386K, the decrease in both kcat and kcat/Km compared with the wild-type enzyme was more pronounced with C5 dicarboxylic substrates (glutamate and 2-oxoglutarate) than with C4 dicarboxylic substrates (aspartate and oxalacetate). The kcat/Km value of AspAT R386K but not of AspAT R292K toward C6 dicarboxylic substrates (2-aminoadipate and 2-oxoadipate) was significantly lower than that toward C5 dicarboxylic substrates. The activity of all mutant enzymes toward alanine and pyruvate was decreased, while kcat/Km of AspAT R292K toward norleucine and 2-oxocaproic acid was even higher than that of the wild-type enzyme.

Table III. Kinetic parameters for transamination half-reactions of wild-type and mutant AspATs with dicarboxylic, monocarboxylic, and aromatic 2-oxo acids

Values were determined under single turnover conditions in 50 mM 4-methylmorpholine, pH 7.5, at 25 °C with 9 µM subunit concentration by Lineweaver-Burk analysis of the rate of the increase in A360 (see "Experimental Procedures"). The concentration ranges of the tested 2-oxo acids were 0.1-10 mM for oxalacetate; 0.5-20 mM for 2-oxoglutarate and 2-oxoadipate; 0.1-50 mM for pyruvate, 0.1-20 mM for 2-oxocaproic acid, and 0.1-5 mM for phenylpyruvate.

Substrates AspAT wild type
AspAT R292K
AspAT R386K
AspAT R292K/R386K
kcat Km kcat/Km kcat Km kcat/Km kcat Km kcat/Km kcat km kcat/Km

s-1 mM M-1 s-1 s-1 mM M-1 s-1 s-1 mM M-1 s-1 s-1 mM M-1 s-1
Oxalacetate 750 0.038a 2 × 107a 9.6b 1.7 5.6 × 103 109 4.5 2.4 × 104 0.06 1.2 1
2-Oxoglutarate 500a 0.83a 6 × 105a 0.3b 9 33.3 0.9b 2.6 346 0.002 15.2 0.13
2-Oxoadipate 0.14b 0.6 233 0.3b 1.8 166 0.002 1 2 0.004 7.8 0.50
Pyruvate 0.10 12.5 8 0.002 31 0.7 0.03 10 3 0.004 31 0.13
2-Oxocaproic acid 0.07 7.3 9.3 0.09 4.2 21 0.001 20 0.05 0.003 52 0.05
Phenylpyruvate c c 1.3 × 103b c c 700b c c 0.7 c c 0.50

a Ref. 7.
b Determined by stopped-flow technique.
c Saturation was not apparent within the concentration range tested. kcat/Km values were obtained by linear least squares regression of the data.

The R386K substitution decreased the catalytic efficiency toward aromatic amino acids by 1-4 orders of magnitude. In contrast, the kcat/Km values of AspAT R292K for aromatic amino acids were only slightly lower than that of the wild-type enzyme and thus higher by 1-2 orders of magnitude than that toward L-aspartate.

Changes in the Binding of Dicarboxylic Inhibitors

The affinity of the mutant enzymes for dicarboxylic substrate analogs of varying length was compared with that of the wild-type enzyme (Table IV). Dicarboxylic acids bind noncovalently to the enzyme and increase the pK'a of the internal aldimine, thereby inducing, at pH 7.5, the conversion of the unprotonated species (lambda max 360 nm) to the protonated species (lambda max 430 nm), thus allowing spectrophotometric determination of their dissociation constants (see "Experimental Procedures"). All aliphatic inhibitors (C4 to C6) with Kd values in the millimolar range in the case of the wild-type enzyme, were bound by all mutant enzymes with a Kd value that was higher by 1 order of magnitude and corresponded to the Kd values of the wild-type enzyme for the too short (C3) and the too long (C7) inhibitors. All enzyme forms showed Kd values >100 mM for monocarboxylic inhibitors (butyrate and n-valerianate). Apparently, the hydrocarbon chain contributes only insignificantly to the binding of the inhibitors. o-Phthalate behaves like C4 to C6 aliphatic inhibitors. m-Phthalate, however, is the only inhibitor that is bound more tightly by the mutant enzymes than by the wild-type enzyme. m-Phthalate with its fixed conformation may be assumed to interact more strongly with the more flexible lysine residue than with the arginine residue, the side chain of which is fixed by multiple interactions of its guanidinium moiety (see Introduction).

Table IV. Dissociation equilibrium constants for dicarboxylic competitive inhibitors of wild-type and mutant AspATs

The values were determined by spectrophotometric titration in 10 mM Tris-acetate, pH 8.0, at 25 °C (see "Experimental Procedures"). The enzyme subunit concentration was 9 µM.

Inhibitors Kd
AspAT wild-type AspAT R292K AspAT R386K AspAT R292K/R386K

mM
Malonate (C3) 50 100 50 100
Maleate (C4) 5.3 51 48 49
Succinate (C4) 2.5 34 48 50
Glutarate (C5) 4.9 21 50 50
Adipate (C6) 4.9 49 49 48
Pimelate (C7) 50 50 50 45
o-Phthalate 5.1 50 51 60
m-Phthalate 4.0 0.9 0.5 0.6


DISCUSSION

Arg right-arrow Lys substitutions are generally considered conservative alterations in protein structure. In AspAT R292K, AspAT R386K, and the double-mutant enzyme, absorption and CD spectra as well as determination of the pKa' value of the internal aldimine indeed did not reveal changes in the active-site geometry. Nevertheless, both single mutations reduced the catalytic efficiency (kcat/Km) for transamination of C4 and C5 dicarboxylic substrates by at least 3 orders of magnitude (Table I). Similar results with AspAT R386K have been reported previously (7).2 A decrease by 6 orders of magnitude was brought about by the double mutation. Substitution of Arg292 with aspartate (26), valine, and leucine (27) has been found to have the same effect. Replacement of Arg386 with tyrosine, phenylalanine (8), or alanine (14) reduced the catalytic efficiency by 4 orders of magnitude. Thus, irrespective of whether Arg292 or Arg386 is substituted and irrespective of the nature of the new side chain, the catalytic efficiency is decreased by at least 3 orders of magnitude.

On the basis of chemical modification studies, it was suggested early on that anionic substrates of enzymes are bound by arginine residues (28). In the case of AspAT, determination of the crystal structure of enzyme-substrate analog complexes (3-6) and of enzymic reaction intermediates (29) has confirmed this prediction. The preference for arginine in AspAT and many other enzymes may be explained by the peculiar strength of the guanidinium-carboxylate interaction due to the resonance-stabilized ion pair underlying the two hydrogen bonds that can be formed (30, 31). Moreover, arginine is a poor proton donor because of resonance stabilization and hence would probably not function as a general acid catalyst. Evolutionary selection of arginine thus minimizes nonspecific hydrolysis of substrates (28). The greater number of possible hydrogen bonds not only with carboxylate groups of the substrates but also with other polar active-site residues endows the guanidinium-carboxylate interaction with a more strictly defined geometry than could be achieved with lysine. The crucial role of arginine-carboxylate interactions, both for substrate binding and efficient catalysis, is borne out by our results.

Wild-type AspAT shows an inverse relationship between kcat/Km values and the side chain length of dicarboxylic amino acids (Table I), as has also been shown in another study (32). The side chain of lysine is shorter than that of arginine, the Calpha -N distance being 5.64 and 6.50 Å, respectively. However, the Arg right-arrow Lys substitution cannot be compensated by longer dicarboxylic substrates. Neither of the substituted arginine residues directly participates in the covalency changes catalyzed by AspAT. Apparently, the loss in enzymic activity is due to modes of binding of the substrates that do not allow the catalytic apparatus to become fully effective. Indeed, all Arg right-arrow Lys mutant AspATs show a decrease in catalytic competence with increasing length of the dicarboxylic substrates, although the binding of dicarboxylic reversible inhibitors is independent of their length (Table IV). Arginine residues that are responsible for the formation of catalytically competent enzyme-substrate complexes and cannot be replaced by lysine without substantial loss in catalytic activity have also been found in enzymes other than AspAT (33-37).

Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long-chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverge. Arg292 binds the distal carboxylate group of dicarboxylic substrates, and its substitution hardly impairs the catalytic competence toward monocarboxylic and aromatic substrates. In contrast, substitution of Arg386, which binds the proximal carboxylate group, decreases the activity toward all types of substrates (Tables I and III).

The introduction of a lysine residue at position 292, which is situated on the re face of the coenzyme-substrate adduct (Fig. 1), increases the rate of racemization of L-aspartate, L-glutamate, and L-alanine 2-3-fold (Table II). A somewhat greater effect was observed when Trp140 on the re face of PLP was replaced by histidine (Table V; Ref. 13). Reprotonation of the coenzyme-substrate adduct at Calpha from the re instead of the si side is a rare event in wild-type AspAT due to the absence of polar residues at the re face and to the almost total exclusion of water in the closed conformation of the substrate-liganded enzyme (24, 38). Conceivably, the R292K substitution interferes with the substrate-induced closure of the active site.

Table V. PLP-dependent catalytic activities of AspAT mutants


Mutation kcat (mutant)/kcat (wild type)
Transamination (L-aspartate + 2-oxoglutarate)  beta -Decarboxylation (L-aspartate) Racemization (L-aspartate) Source

I17H 0.2 a BDb Ref. 13
W140H 0.2 1 7c Ref. 13
Y225R/R386A 0.001 650 BD Ref. 14
R292K 0.002 15 3 This paper
R386K 0.01 3 1 This paper
R292K/R386K 0.0001 3 1 This paper

a kcat = 8 × 10-5 s-1; wild-type mitochondrial AspAT has no detectable beta -decarboxylase activity.
b BD, activity below detection level.
c L-Alanine as substrate.

The R292K mutation increases not only the racemase activity but also enhances the L-aspartate beta -decarboxylase activity 15-fold in comparison with the wild-type enzyme (Table II). For efficient beta -decarboxylation, a proton donor has to operate in close proximity to the position of the leaving carboxylic group (25). The accelerated beta -decarboxylation observed with AspAT R292K may be explained, at least in part, by the presence of Lys292, which is a much better proton donor than arginine and might, assisted by an intervening water molecule, protonate the carbanion intermediate 5 (Scheme 1) produced by beta -decarboxylation. The reaction pathways of transamination, beta -decarboxylation, and racemization diverge only after deprotonation at Calpha of the external aldimine 2 has formed the quinonoid intermediate 3. In transamination, reprotonation of this intermediate at C4' produces the ketimine intermediate 4. In the wild-type enzyme, despite its much higher transaminase activity, both removal of CO2 from Cbeta and reprotonation at Calpha from the re side to give the aldimine intermediate 6 are slower than in the mutant enzymes.


Scheme 1. Intermediates in the reactions of aspartate catalyzed by wild-type and mutant AspATs.
[View Larger Version of this Image (13K GIF file)]

In conclusion, neither of the two substrate-binding arginine residues of E. coli AspAT can be replaced by lysine without a marked decrease in the affinity for dicarboxylic substrates and an even larger decrease in the catalytic activity toward these substrates. The exact positioning of the substrate moiety, the additional hydrogen bonds of the guanidinium group to other active-site residues, and the specific electrostatic potential due to the guanidinium groups are all factors that conceivably contribute to efficient catalysis. The present data and earlier results (Table V) provide unequivocal experimental evidence that the reaction specificity of PLP-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing the occurrence of potential side reactions. Most changes in the delicately poised catalytic machinery of AspAT have indeed resulted not only in a decrease in rate of the specific reaction, i.e. transamination, but also in an acceleration of the side reactions, i.e. beta -decarboxylation and racemization.


FOOTNOTES

*   This work was supported in part by an Italian National Research Council Bilateral Project, the Swiss National Science Foundation Grant 31-45940.95, and the COST ACTION-D7 Program of the European Cooperation in the Field of Scientific and Technical Research.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.
1   The abbreviations used are: PLP, pyridoxal 5'-phosphate; AspAT, aspartate aminotransferase; PMP, pyridoxamine 5'-phosphate; Mes, 4-morpholineethanesulfonic acid; Ches, 2-(cyclohexylamino)ethanesulfonic acid.
2   Hayashi, H., Kuramitsu, S., Inoue, Y., Tanase, S., Morino, Y., and Kagamiyama, H. (1989) Posters 357 and 358 presented at Protein Engineering '89, August 20-25, (1989) (Protein Eng. 2, 357-358).

ACKNOWLEDGEMENTS

We thank Dr. Alberto Boffi and Peter Latal for valuable assistance in performing stopped-flow kinetic measurements.


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