(Received for publication, April 29, 1996, and in revised form, August 19, 1996)
From the Laboratory of Cellular Biochemistry, Facultés Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium, and the § Department of Biochemistry, Physiology and Microbiology, Rijksuniversiteit, 35 Ledeganckstraat, B-9000 Gent, Belgium
L-Alanine dehydrogenase from
Bacillus subtilis was inactivated with two different
lysine-directed chemical reagents, i.e. 2,4,6-trinitrobenzenesulfonic acid and N-succinimidyl
3-(2-pyridyldithio)propionate. In both cases, the inactivation followed
pseudo first-order kinetics, with a 1:1 stoichiometric ratio between
the reagent and the enzyme subunits. Partial protection of the
active site from inactivation could be obtained by each of the
substrates, NADH or pyruvate, but complete protection could only be
achieved in the presence of the ternary complex
E·NADH·pyruvate. The nucleotide analogue of NADH,
5-(p-(fluorosulfonyl)benzoyl)adenosine was also used for affinity labeling of the enzyme active site.
Differential peptide mapping, performed both in the presence and in the absence of the substrates, followed by reversed phase high performance liquid chromatography separation, diode-array analysis, mass spectrometry, and N-terminal sequencing of the resulting peptides, allowed the identification of lysine 74 in the active site of the enzyme. This residue, which is conserved among all L-alanine dehydrogenases, is most likely the residue previously postulated to be necessary for the binding of pyruvate in the active site.
Surprisingly, this residue and the surrounding conserved residues are not found in amino acid dehydrogenases like glutamate, leucine, phenylalanine, or valine dehydrogenases, suggesting that A-stereospecific amino acid dehydrogenases such as L-alanine dehydrogenase could have evolved apart from the B-stereospecific amino acid dehydrogenases.
L-Alanine dehydrogenase (AlaDH1; EC 1.4.1.1) first identified by Wiame and Piérard (1), and later purified from Bacillus subtilis by Yoshida and Freese (2-4), catalyzes the reversible oxidative deamination of L-alanine to pyruvate and ammonium (Reaction R1).
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The amino acid sequence of AlaDH has been first determined from the
strains Bacillus sphaericus and Bacillus
stearothermophilus (11). The sequence fingerprint characteristic
of the Rossmann's fold responsible for the nucleotide
binding (12, 13) has been recognized. Alignment of AlaDH sequences with
other amino acid dehydrogenases like glutamate dehydrogenase,
leucine dehydrogenase, or phenylalanine dehydrogenase, and with
hydroxyacid dehydrogenases like lactate dehydrogenase or malate
dehydrogenase led Kuroda et al. (11) to propose His-153 and
Lys-156 to be part of the catalytic site. However, the availability of
a third AlaDH sequence, obtained from Mycobacterium
tuberculosis (14) did not support this hypothesis and stressed the
need to investigate the composition of the active site (15).
Sequence comparisons between AlaDH and other proteins available in data banks have shown high similarities between AlaDH and the N-terminal part of pyridine nucleotide transhydrogenase (15, 16), for which a three-dimensional model of the NAD+ binding site has been proposed (17). However, no convincing sequence resemblance between AlaDH and the other enzymes of the amino acid dehydrogenase superfamily has been found. In that respect, AlaDH constitutes an exception from the other amino acid dehydrogenases, like glutamate dehydrogenase, whose three-dimensional structure has been elucidated (18), or leucine dehydrogenase, phenylalanine dehydrogenase, and valine dehydrogenase, which have been shown to share sequence and structure similarities with glutamate dehydrogenase (19, 20). These similarities include an identical B-type stereospecificity with respect to NAD+ (21-23) and a common organization of the residues implicated in the catalytic chemistry (19).
We have much information on the active-site structure of B-stereospecific amino acid dehydrogenases, obtained by chemical modification studies (24-28), by genetic engineering (27, 29-32), and from x-ray crystallographic data (18, 33, 34). However, very scarce information is available for the A-stereospecific amino acid dehydrogenases such as alanine dehydrogenase. Little is known about the residues that might be involved in substrate binding and catalysis (8, 35, 36), and no catalytic amino acid residue has been identified. Recently, the amino acid sequence from B. subtilis alanine dehydrogenase has become available (6), allowing the interpretation of chemical modification studies performed on this enzyme.
In this paper, we provide evidence that Lys-74 of B. subtilis alanine dehydrogenase is located at the active site of the enzyme. That residue is conserved among all alanine dehydrogenases sequenced so far and is likely the lysine residue that is required for the binding of pyruvate during the catalytic reaction.
L-Alanine dehydrogenase from B. subtilis purchased from Sigma was desalted before
use on a 15 × 2.5-cm Ultrogel AcA44 column (IBF Biotechnics,
Villeneuve-la-Garenne, France) equilibrated in 100 mM
NaH2PO4, pH 7.5. The substrates NADH and
pyruvate and the chemical reagents 2,4,6-trinitrobenzenesulfonic acid
(TNBS), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP),
and 5-(p-(fluorosulfonyl)benzoyl) adenosine (FSBA) were
also obtained from Sigma. Endoproteinase Glu-C
sequencing grade was from Boehringer (Mannheim, Germany). All other
reagents were from Merck (Darmstadt, Germany), except Tris, guanidine
HCl, and trifluoroacetic acid, which were from Acros Chimica (Beerse,
Belgium), and acetonitrile, which was from Carlo Erba (Milano,
Italy).
Alanine dehydrogenase activity was assayed for alanine synthesis according to Yoshida et al. (3). In typical experiments, the assay mixture (0.6 ml) contained 0.5 mM NADH, 2 mM pyruvate, and 100 mM NH4Cl in 100 mM Tris-HCl, pH 8.5. The reaction was started by the addition of 1-2 µg of protein to the mixture. The assay was carried out at 20 °C by recording the decrease in absorbance of NADH at 340 nm, with a Kontron 930 variable wavelength spectrophotometer. Protein assay was performed by the Folin reagent method (37), using bovine serum albumin as standard. When the samples contained Tris-HCl as buffer, the protein concentration was determined by the dye binding assay (38), using the kit provided by Bio-Rad (München, Germany).
Chemical Modification with TNBSAlaDH was inactivated by incubating the enzyme (1 mg/ml) in the dark, at 40 °C, in the presence of varying concentrations of TNBS (39) (stock solutions diluted in ice-cold distilled water), in 100 mM NaH2PO4, pH 8.0. Aliquots (2 µl) were removed at regular time intervals and mixed with the assay solution to measure residual activity. Control experiment showed that the enzyme did not loose any activity in those conditions when TNBS was omitted.
Chemical Modification with SPDPInactivation by SPDP (40)
was also performed at a protein concentration of 1 mg/ml, in 100 mM NaH2PO4, but the pH was adjusted to 7.5 and the experiment was carried out at 20 °C during 10 min. SPDP at varying concentrations was dissolved in ethanol before addition
to the protein solution. Residual activity was also assayed at
different times as indicated above. A control experiment showed that
the enzyme did not lose any activity when the experiment was performed
in the same conditions as without SPDP. In the pH conditions mentioned,
chemical modification by SPDP is known to be specific for primary amino
groups (-NH2 group of lysines or N-terminal
NH2-group of the protein) rather than for cysteines (40).
However, we performed a control experiment by incubating SPDP-inactivated AlaDH in 25 mM
NaH2PO4, pH 7.8, in the presence of 5 mM dithiothreitol at 20 °C (45 min), in order to check
the possible reaction of SPDP with a putative active-site cysteine of
the enzyme.
AlaDH (1 mg/ml) was inactivated by FSBA (41) at varying reagent concentrations (stock solution dissolved in dimethyl sulfoxide) in 100 mM NaH2PO4, pH 7.5, at 40 °C (in the dark), and the activity was measured at time intervals. Also here, the enzyme showed no loss of activity in the incubation conditions when FSBA was omitted.
Protection StudiesThe ability of substrates to protect the active site of the enzyme from inactivation was estimated by testing the effect of NADH and/or pyruvate. Except when indicated otherwise in the text, alanine dehydrogenase was at 1 mg/ml and NADH·pyruvate was added at a final concentration of 0.44 mM (100 × molar excess with respect to enzyme concentration) and 44 mM (10,000 × molar excess), respectively, before the start of the chemical modification.
Proteolysis of Chemically Modified Alanine DehydrogenaseSeveral samples of alanine dehydrogenase (1.2-2.0 mg; 1 mg/ml; 4.4 µM) were modified separately with the chemical reagents TNBS, SPDP, or FSBA in the above mentioned conditions. Reagent concentrations and incubation times were, respectively, 0.88 mM (60 min), 0.44 mM (10 min), and 1.76 mM (30 min) for TNBS, SPDP, and FSBA modification. After the reaction, AlaDH was separated from the chemicals by chromatography on an Ultrogel AcA44 column (2.5 cm × 15 cm; IBF Biotechnics, Villeneuve-la-Garenne, France) equilibrated with 25 mM NaH2PO4, pH 7.8. The enzyme was then reconcentrated over a PM10 ultrafiltration membrane in a Centricon concentration system from Amicon (Lexington, MA). Protein concentration was adjusted to 1.2 mg/ml, and 6 M guanidine HCl in 25 mM NaH2PO4, pH 7.8, was added to the solution at a ratio of 20:100 (v/v). Digestion was achieved using endoproteinase Glu-C (42) (1 mg/ml) at a ratio of 1:50 (w/w) relative to the enzyme, during 16 h at 25 °C. The digest (400 µl to 1 ml) was then analyzed on a reversed phase HPLC. A control experiment with native AlaDH was performed in parallel, in exactly the same conditions.
Separation and Detection of PeptidesPeptides resulting
from enzymatic cleavage were separated by reversed phase HPLC on a
C18 Nucleosil column (Macherey-Nagel, Düren,
Germany), using the HP1090 HPLC system (Hewlett-Packard, Palo Alto, CA)
equipped with a 1040M Series II multiple wavelength diode-array
detector. Samples (1 mg/ml, 500 µl) were filtered through a 0.2-µm
membrane Nylon Acrodisc (Gelman Sciences, Ann Arbor, MI) before
injection onto the column previously equilibrated with solvent A. The
gradient used was 0-20% B for 5 min (elution of guanidine HCl and
buffer), followed by 20-80% B for 72 min (elution of the peptides),
and a wash by 100% B during 15 min. Solvent A was trifluoroacetic acid
0.1% (v/v) in water, solvent B was trifluoroacetic acid 0.1% (v/v) in
water-acetonitrile (20:80). Elution was carried out at a flow rate of 1 ml/min, with the multiple wavelength detector, respectively, set at
220/280/346, 220/280/304, and 220/280/259 nm for TNBS-, SPDP-, and
FSBA-labeled samples. Control experiments were also performed by
injecting the reagents alone under the same chromatographic conditions.
Eluted peptides were collected and either frozen at 70 °C or
lyophilized for further analysis.
When necessary, peptides of interest were further purified by HPLC under the same chromatographic conditions, except that a linear gradient of 0.25% increase of solvent B/min was used. Alternatively, peptides were repurified on a C18 column from Pharmacia (Uppsala, Sweden) using the Pharmacia Smart system with a linear gradient of 0-70% solvent B over 45 min.
Mass Analysis of Peptides and Amino Acid Sequence DeterminationPeptides of interest were dissolved in acetonitrile/1% acetic acid in water (50:50) and their mass determined in a VG Platform electrospray ionization mass spectrometer (Fisons Instruments, Manchester, United Kingdom). The amino acid sequence of peptides was determined on a model 475A peptide sequencer (Applied Biosystems, Foster City, CA) equipped with on-line phenylthiohydantoin-derivative analyzer.
TNBS and
N-hydroxysuccinimide esters like SPDP are known to be highly
selective reagents at pH 7.5-8.0 for the modification of lysine
residues (-NH2 group of the side chain) and/or N
terminus of proteins (39, 40). Incubation of L-alanine
dehydrogenase with varying concentrations of TNBS and SPDP resulted in
a time-dependent loss of enzyme activity, suggesting the
modification of a primary amino group located at or near the enzyme
active site. Plots of the logarithm of remaining activity
versus time at different reagent concentrations indicated in
each case pseudo first-order kinetics (Figs.
1A and 2A). A
straight line was also observed for the plot of pseudo first-order rate
constants versus reagent concentration (Figs. 1B
and 2B), indicating that the chemical modification is the
result of a simple bimolecular reaction. The second-order rate
constants (kinact) obtained for the modification
by TNBS and SPDP were 0.57 and 60.6 M
1
s
1, respectively, and show a much higher reactivity of
SPDP compared to TNBS toward the active-site lysine residue. Plotting
log kinact versus log of reagent
concentration, according to Levy et al. (43), yields an
apparent reaction order of 0.88 and 1.00 for TNBS and SPDP,
respectively, indicating that inactivation results from the reaction of
approximately 1 mol of reagent with 1 mol of enzyme subunit (Figs.
1C and 2C). This observation is in agreement with
the result obtained for the stoichiometry of the reaction with SPDP,
which indicates that the modification of no more than 1.4 lysine
residues/monomer is required for the complete inactivation of AlaDH
(Fig. 3).
SPDP is a bifunctional reagent that can also react with cysteine residues to form a disulfide link (40). In order to estimate the possible involvement of cysteine reaction, we incubated SPDP-inactivated AlaDH in the presence of 5 mM dithiothreitol (45 min, 20 °C) to determine whether reduction of the enzyme could restore its activity. Only 17.5% of activity could be restored by this treatment, assessing that inactivation with SPDP was mainly the result of modification of a lysine rather than a cysteine residue.
Chemical Modification with FSBAIn a third series of
experiments, we used the structural analog of NADH, FSBA, for specific
modification of the alanine dehydrogenase cofactor binding site. This
reagent has proved to be appropriate for active-site affinity labeling
of other dehydrogenases like malate dehydrogenase, 20-hydroxysteroid
dehydrogenase, or 17
-estradiol dehydrogenase (41). The time course
of AlaDH inactivation in the presence of FSBA followed pseudo
first-order kinetics (Fig. 4A), but, in this
case, complete inactivation of the enzyme could not be obtained even at
high reagent concentration. The activity declined to a non-zero value
after a relatively long period (60 min of incubation). A minimum
residual activity of about 25% was obtained with 10 mM
FSBA (Fig. 5A). The rate of inactivation
increased according to the initial FSBA concentration, showing typical
saturation kinetics. This is observed in the plot of pseudo first-order
rate constants kinact versus FSBA
concentration (Fig. 4B) and in the plot of log
kinact versus log FSBA concentration
(Fig. 4C), where a deviation from linearity is observed at
high reagent concentrations. Fitting straight lines by linear
regression on the linear portion of data points gave values of 5.47 × 10
2 M
1 s
1 and
0.76 for the second-order rate constant and the apparent order of
reaction, respectively, but a polynomial second-order curve best fitted
the data points in Fig. 4B with a correlation coefficient of
R2 = 1.00. These results clearly show that
chemical modification of AlaDH with FSBA does not follow a simple
bimolecular mechanism as observed for TNBS or SPDP but proceeds through
a two-step reaction. This is in agreement with a mechanism where FSBA
first binds to the adenosine binding site of the enzyme to form a
reversible non-covalent complex (inhibition) and subsequently
reacts in an irreversible covalent way with an amino acid residue of
the active site (inactivation) (Reaction R2).
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(Eq. 1) |
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(Eq. 2) |
Protection Studies
In order to assess that the chemical
modification by TNBS, SPDP, and FSBA is active-site directed, we tested
the ability of substrates to protect the enzyme active site from
inactivation (Fig. 6). The mechanism of reaction of
AlaDH is known to be ordered with first the binding of NADH, followed
by pyruvate and ammonium (7). We could not assay the protection by
L-alanine or NH4Cl since they can react with
the chemical reagents used. We tested the protecting effect of NADH,
pyruvate, and pyruvate analogues. In the case of TNBS and SPDP, only
little or no protecting effect was observed when the substrates NADH or
pyruvate were used alone. However, the kinact
was dramatically reduced when NADH and pyruvate were used together.
This result clearly shows that the lysine residue reacting with these
chemicals is located in the active site of the enzyme and can only be
protected in the presence of the ternary complex
E·NADH·pyruvate. The inability of NADH alone to protect
the enzyme from inactivation suggest that it does not occur at the NADH
binding site itself but more likely at or near the pyruvate binding
site of the enzyme as the presence of pyruvate is also required for
good protection. The inability of pyruvate alone to protect from
inactivation can be understood by the ordered mechanism of reaction in
which pyruvate can only bind to the enzyme when NADH is first fixed at
the active site.
We then tested the ability of several pyruvate analogues in combination
with NADH to protect AlaDH from inactivation, in order to see whether
there was a correlation between the ability of these compounds to act
as substrates and to afford protection against the lysine modification
(Table I). The results show that increasing the length
of the aliphatic chain of ketoacids from methyl (in pyruvate) to propyl
(in -ketovalerate) gradually reduces the protecting effect of the
substrates. Replacing the methyl group of pyruvate by a hydrogen (in
glyoxylate) or a carboxylic acid chain (in the dicarboxylic acid
series) completely abolishes the protecting effect, except for
oxalacetate, which exhibits a strikingly high protecting effect. These
results confirm the presence of the lysine residue near the pyruvate
binding site since oxalacetate is a good substrate for the enzyme, the
product of the reaction being D-aspartate and not
L-aspartate as it could be expected (data not shown). This
is in contrast to the other dicarboxylic acids which are not accepted
as substrates. Finally, replacing the methyl group of pyruvate by
-NH2 in oxamate gave some protection against inactivation.
This is also in agreement with the fact that oxamate is an isosteric
and isoelectronic substrate analogue of pyruvate and is known to be a
competitive inhibitor of alanine dehydrogenase (7).
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Similar results for substrate protection were obtained for FSBA inactivation, except that here a greater effect was obtained for the protection by NADH alone (Fig. 6C). This observation is in agreement with the fact that, because of its structural similarity to NADH, FSBA is expected to bind at the NADH binding site before its covalent reaction with an amino acid of the active site. Nevertheless, here again the simultaneous presence of both NADH and pyruvate is required for a complete protection against inactivation.
Identification of Modified Amino Acid ResiduesL-Alanine dehydrogenase was chemically modified using TNBS, SPDP, and FSBA as described under "Experimental Procedures." Samples were passed through a desalting column, reconcentrated, and submitted to proteolysis using endoproteinase Glu-C (42). For TNBS and SPDP inactivation, experiments were carried out in parallel both in the presence of NADH and pyruvate (protection of the whole active site) or in the presence of NADH alone (protection of the NADH binding site only).
The HPLC profiles obtained for TNBS-modified alanine dehydrogenase are
presented in Fig. 7. The comparison of the profiles shows that the peak eluting at 32.5 min disappears when pyruvate is
absent from the active site (Fig. 7, A and B,
peak I), while another peak eluting at 46.0 min increases
(peak II). This increase is particularly obvious at 346 nm
(Fig. 7D), which is a specific wavelength for TNBS chemical
modification. Diode-array detection analysis of these compounds
indicated that peak II was labeled with TNBS, while peak I was not, as
shown by its characteristic absorbance profile with a maximum at 346 and 420 nm (Fig. 8A). Peptides from peaks I
and II were collected and submitted to mass determination and to
N-terminal sequencing (over 6-10 residues) by automated Edman
degradation. For the peak eluting at 32.5 min, the sequence MVMKVK
could be identified (Table II). This result, together
with the mass determination obtained (Mr = 2286.8) indicates that peptide I corresponds to Met-69 to Lys-86 (Table
III). Interestingly, a similar sequence was found in the
peptidic fragments identified in peak II, where the mass observed for
one of these two fragments was consistent with the TNBS labeling of the
peptide Met-69-Lys-86 (Table II, fragment IIa). Unfortunately, only a
few phenylthiohydantoin-derivatives of this fragment could be
identified due to the low amount of material available, but they all
corresponded to the expected sequence. Another peptidic fragment (Table
II, fragment IIb), starting with a proline corresponding to Pro-76 in
the AlaDH sequence, contaminated peak II.
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The HPLC profile of the peptides obtained after chemical modification
of alanine dehydrogenase with SPDP is presented in Fig. 9. Similarly to the results obtained in Fig. 7, the peak
eluting at Rt = 32.5 min (peak I) decreases when
pyruvate is absent from the active site, while another peak increases,
which absorbs at 304 nm (Fig. 8B) and elutes at
Rt = 39.8 min (Fig. 9, peak III). This
result suggests that the SPDP chemical labeling modified the same
peptidic fragment of alanine dehydrogenase as the one modified with
TNBS, i.e. Met-69 to Lys-86. This was also confirmed by the
mass analysis and sequence determination of the peptides contained in
peak III (Table II), where masses of 2484.8 and 2374.2 are in agreement
with the SPDP-labeling of peptide I (in the oxidized and reduced
states), while the mass of 3912.1 is consistent with a longer
SPDP-labeled fragment from Met-69 to Glu-99. The N-terminal
sequencing of the peptide from peak III gave the consensus
sequence MVMKV*EP, with an unidentified phenylthiohydantoin-derivative at the 6th cycle, corresponding to
Lys-74 in L-alanine dehydrogenase (Table II).
The elution pattern of the peptides resulting from endoproteinase Glu-C
digest of FSBA-labeled alanine dehydrogenase (Fig. 10)
was obtained similarly to those obtained after the TNBS and SPDP
chemical modification, except that the experiment was performed both in
the presence of NADH and pyruvate (protection of the whole active site)
and without any substrate (no protection). This reagent, which
potentially can react with several residues of the active site, also
reacted with the same peptidic fragment as indicated by the decrease of
peak I when the substrates were omitted (Fig. 10, A and
B). Peak IV appearing in those conditions highly absorbs at
259 nm (Fig. 8C and 10D) and gave the sequence
MVMKV*EPLP, with masses of 2721.0 and 3883.0 (Table II).
L-Alanine dehydrogenase from B. subtilis was completely inactivated using TNBS and SPDP. In both cases, the inactivation of the enzyme was the result of a simple bimolecular reaction, with the modification of about one lysine residue/monomer. This result suggests the presence of an essential lysine residue at or near the active site of the enzyme. A different pattern of inactivation was obtained for the modification of AlaDH with the structural analogue of NADH, i.e. FSBA. In this case, saturation kinetics were obtained, indicating a two-step mechanism where FSBA binds to the NADH binding site of the enzyme before irreversible chemical modification of an active-site residue. Results obtained for the chemical modification of the enzyme in the presence of the substrates indicate that NADH or pyruvate alone do not allow a good protection, and that the enzyme can only be effectively protected when the ternary complex E·NADH·pyruvate is formed. Given the ordered mechanism of AlaDH where NADH binds before pyruvate (7), the fact that both NADH and pyruvate are required for a good protection of the active site suggests that the chemical modification occurs at the pyruvate binding site, rather than at the NADH binding site.
Differential peptide mapping, both in the presence and in the absence of the substrates, and monitoring at a wavelength specific for the label, allowed the identification of active-site peptide fragments. For both TNBS and SPDP, the same peptidic fragment from Met-69 to Lys-86 was found to be labeled, with a concomitant retention time modification and increase of absorbance of the corresponding peak. Sequence comparisons of the isolated fragments with the known sequence of B. subtilis alanine dehydrogenase (6) indicated that the cleavage between Glu-68 and Met-69 is consistent with the expected cleavage specificity of endoproteinase Glu-C, i.e. the cleavage after Glu or Asp residues (42). On the other hand, the absence of cleavage after Glu-75, Glu-79, and Glu-80 is probably due to the presence of two proline residues, respectively, at position 76 and 78 of the sequence. These residues are known to form secondary structures that often prevent the recognition of proteases with their substrates. More surprising was the aspecific cleavage of endoproteinase Glu-C after a lysine residue in position 86, which was obtained for several independent experiments. This unusual cleavage after a lysine residue is not in the list of the several aspecific cleavages that have been reported for endoproteinase Glu-C, for example after Gly and Ala (45), Asn and Tyr (46), or Gln and Ser (47). To our knowledge, cleavage after a lysine had not yet been observed.
Mass and sequence determinations of the isolated peptides were
consistent with the chemical labeling of lysine 74 of the
L-alanine dehydrogenase, implying the presence of this
residue at or near the active site of the enzyme. Interestingly, the
active-site affinity labeling using FSBA also modified the same
Met-69-Lys-86 peptidic fragment at the position of lysine 74, although
FSBA can react with several residues other than lysine. Sequence
alignment of this peptide with the known sequences of B. subtilis AlaDH (6), B. sphaericus (11), B. stearothermophilus (11), M. tuberculosis (14), and
Synechocystis sp. (48) indicates that the modified lysine
residue is conserved among all the alanine dehydrogenases sequenced to
date, and that it is located in an important stretch of five conserved
residues KVKEP from Lys-72 to Pro-76 (Table III). According to the
sequence analysis of the enzyme (15), these residues are most likely
located outside of the NADH binding site, which is supposed to expand
around and after the characteristic
GXGXXG(X17)D motif of the
Rossmann's fold. This observation is in agreement with a
localization of Lys-74 at the pyruvate binding site but in close
vicinity of the NADH binding site, since the same Lys-74 is also
modified by FSBA. The presence of Lys-74 in the NADH binding site
itself is not completely excluded. In this case, the protection
obtained for the enzyme inactivation by TNBS and SPDP, only when both
NADH and pyruvate are present, would implicate that Lys-74 would still be accessible to these chemicals when NADH alone is bound to the active
site, but that a conformational change occurs when pyruvate is bound,
so that only in this situation, Lys-74 would be part of the NADH
binding site. This possibility cannot be ruled out, but all the
arguments presented here above argue against this explanation.
According to Grimshaw et al. (8), a cationic acid group on the enzyme (probably a lysine) is required for effective binding of the substrate and the inhibitors, while another cationic acid group (probably a histidine), acts as an acid-base catalyst of the reaction. In an attempt to locate these residues in the sequence of B. sphaericus and B. stearothermophilus, Kuroda et al. (11) performed sequence comparisons with other amino acid dehydrogenases (glutamate dehydrogenase, phenylalanine dehydrogenase, and leucine dehydrogenase) and with hydroxyacid dehydrogenase, which share some substrate and catalytic features with alanine dehydrogenase (lactate dehydrogenase and malate dehydrogenase). In their conclusions, the authors proposed His-153 and Lys-156 from B. sphaericus to be part of the active site (11, 27). However, the availability of a third alanine dehydrogenase obtained from M. tuberculosis (14) ruled out this hypothesis, since the proposed residues were not conserved in this new sequence (15). The experimental results obtained by us clearly identify Lys-74 as part of the enzyme active site, and support its role in the catalytic mechanism of the L-alanine dehydrogenase.
In a previous paper the sequence of B. sphaericus alanine dehydrogenase was compared with the protein sequences of the Swissprot, GenBank, and EMBL data bases (15). Surprisingly, no other amino acid dehydrogenase or hydroxyacid dehydrogenase was found to be significantly similar to alanine dehydrogenase, but the enzyme was found to be similar to the N-terminal sequence of pyridine nucleotide transhydrogenase, suggesting a similar folding of these two protein segments (15). However, no alanine dehydrogenase activity was detected in M. tuberculosis pyridine nucleotide transhydrogenase (56), suggesting that even if they have a similar structure, their active site is different. In agreement with this active-site difference, pyridine nucleotide transhydrogenase also lacks lysine 74, which was found to be essential for the activity of alanine dehydrogenase (Table III).
L-Alanine dehydrogenases appear as very unique enzymes among the amino acid dehydrogenases. Alanine dehydrogenase has been shown to be a member of A-stereospecific dehydrogenases (10, 57), unlike the other amino acid dehydrogenases studied to date, which are B-stereospecific (21-23). In this paper, we showed that alanine dehydrogenases from B. subtilis posses a lysine at the position 74 that is essential for the enzyme activity, and that this residue is conserved among the other alanine dehydrogenases sequenced so far (Table III). This lysine and the surrounding conserved sequence region are not found in other dehydrogenases. Furthermore, the characteristic active-site motif K(X8)GGXK identified in glutamate, leucine, phenylalanine, and valine dehydrogenases (19, 20) is not found in L-alanine dehydrogenases, suggesting a separate evolution of these two groups of amino acid dehydrogenases. The only similarity of sequence was found with one part of the pyridine nucleotide transhydrogenase, which suggests that L-alanine dehydrogenase, contrary to the B-stereospecific amino acid dehydrogenases, may have evolved along with pyridine nucleotide transhydrogenases rather than with the other dehydrogenases of the amino acid dehydrogenase superfamily.
We gratefully acknowledge John D. Shannon from the University of Virginia for information on nonspecific cleavages by endoproteinase Glu-C.