(Received for publication, June 9, 1997, and in revised form, June 27, 1997)
From the 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
Arg386 and Arg292
of aspartate aminotransferase bind the 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
-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
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.
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
-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).
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.
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
280 = 4.7 × 104 M
1 cm
1
(21).
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 pKSpectral 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 pKa 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.
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.
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
pKa 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
Lys mutant AspATs.
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
kcat 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).
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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.
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 -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
-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
-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.
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.
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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 InhibitorsThe
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
pKa of the internal aldimine,
thereby inducing, at pH 7.5, the conversion of the unprotonated species (
max 360 nm) to the protonated species
(
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).
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Arg 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 C-N distance being 5.64 and 6.50 Å, respectively. However, the Arg
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
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 C 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.
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The R292K mutation increases not only the racemase activity but also
enhances the L-aspartate -decarboxylase activity 15-fold in comparison with the wild-type enzyme (Table II). For efficient
-decarboxylation, a proton donor has to operate in close proximity to the position of the leaving carboxylic group (25). The accelerated
-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
-decarboxylation. The reaction pathways of transamination,
-decarboxylation, and racemization diverge only after deprotonation
at C
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 C
and reprotonation at C
from the
re side to give the aldimine intermediate 6 are slower than in the mutant enzymes.
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. -decarboxylation and racemization.
We thank Dr. Alberto Boffi and Peter Latal for valuable assistance in performing stopped-flow kinetic measurements.