(Received for publication, February 6, 1997)
From the Biochemisches Institut der Universität Zürich, CH-8057 Zürich, Switzerland
Cofactors may be expected to expand the range of
reactions amenable to antibody-assisted catalysis. The biological
importance of pyridoxal 5-phosphate (PLP) as enzymic cofactor in amino
acid metabolism and its catalytic versatility make it an attractive candidate for the generation of cofactor-dependent abzymes.
Here we report an efficient procedure to screen antibodies for
PLP-dependent catalytic activity and detail the spectrum of
catalytic activities found in monoclonal antibodies elicited against
N
-(5
-phosphopyridoxyl)-L-lysine.
This hapten is a nonplanar analog of the planar, resonance-stabilized
coenzyme-substrate adducts formed in the PLP-dependent
reactions of amino acids. The hapten-binding antibodies were screened
for binding of the planar Schiff base formed from PLP and
D- or L-norleucine by competition enzyme-linked immunosorbent assay. The Schiff base (external aldimine) is an obligatory intermediate in all PLP-dependent reactions of
amino acids. This simple, yet highly discriminating screening step
eliminated most of the total 24 hapten-binding antibodies. Three
positive clones bound the Schiff base with L-norleucine,
two preferred that with the D-enantiomer. The positive
clones were assayed for catalysis of Schiff base formation and of the
,
-elimination reaction with the D- and
L-enantiomers of
-chloroalanine. Three antibodies were
found to accelerate aldimine formation, and two of these catalyzed the
PLP-dependent
,
-elimination reaction. One of the
,
-elimination-positive antibodies catalyzed the transamination reaction with hydrophobic D-amino acids and oxoacids
(Gramatikova, S. I., and Christen, P. (1996) J. Biol.
Chem. 271, 30583-30586). All catalytically active antibodies
displayed continuous turnover. No PLP-dependent reactions
other than aldimine formation,
,
-elimination of
-chloroalanine
and transamination were detected. The successive screening steps
plausibly simulate the functional selection pressures having been
operative in the molecular evolution of primordial PLP-dependent protein catalysts to reaction- and
substrate-specific enzymes.
The first catalytic antibodies that became known accelerated
relatively simple transformations; since then the antibody-catalyzed reactions have increased in complexity and degree of difficulty. Possible strategies to expand the catalytic scope of antibodies include
the incorporation of cofactors such as metal ions, heme, thiamine,
flavins, nicotinamide, or pyridoxal into the binding sites of the
antibodies (1). Pyridoxal 5-phosphate
(PLP)1 is probably the most versatile
enzymic cofactor. PLP is required by many enzymes that catalyze a wide
variety of reactions in the metabolism of amino acids, i.e.
transamination, racemization, decarboxylation, aldol cleavage, and
elimination and replacement reactions (2). Several attempts to produce
pyridoxal-dependent catalytic antibodies have been
reported. In the earliest study, a polyclonal antiserum specific for
the reduced Schiff base formed from PLP and
3
-amino-L-tyrosine was prepared. The antibodies slightly
enhanced the rate of the PLP-catalyzed transamination of
L-tyrosine (3-5). A monoclonal antibody against the
reduced aldimine of pyridoxal and 4
-nitro-L-phenylalanine
accelerated aldimine formation between 5
-deoxypyridoxal and
4
-nitro-D-phenylalanine but did not catalyze any further
reactions (6). Catalysis of Schiff base formation was also observed
with a polyclonal antiserum generated against the reduced Schiff base
of pyridoxal and D- and L-phenylalanine
(7).
The Schiff base 4 generated by condensation of PLP
1 and amino acid 2 (Fig. 1) is the
first detectable intermediate common to all nonenzymic and enzymic
PLP-dependent reactions of amino acids. In the enzymic
reactions, the Schiff base is produced by transimination; the amino
group of the substrate replaces the -amino group of the active-site
lysine residue which covalently binds PLP in all B6
enzymes. The multiple possibilities for further reactions of the
coenzyme-substrate aldimine 4 give rise to the diverse
PLP-dependent transformations of amino acids (Scheme
1). Reduction of the imine double bond of the aldimine by sodium borohydride provides a stable link between the coenzyme and
the amino acid. The C
-nitrogen linkage of the resulting
phosphopyridoxyl amino acids 5 (Fig. 1) is similar to that
in the tetrahedral carbinolamine transition state 3 leading
to Schiff base formation (8). Phosphopyridoxyl amino acids bind with
high affinity to apoenzymes (5, 9, 10) and include all groups important
for catalysis with the exception of the imine double bond ensuring the
planarity of the Schiff base (Fig. 1). Formation of the planar Schiff
base is, however, a prerequisite for the catalytic efficacy of PLP
which is due to the electron-withdrawing effect exerted on C
by the
positively charged pyridine nitrogen atom and is mediated through the
extensive resonance system of the pyridine ring and the imine double
bond.
In a renewed attempt to obtain PLP-dependent antibody
catalysts, we used, as in the previous studies by other laboratories (3-7), a reduced Schiff base as hapten for immunization. The structural disadvantage of this transition state analog was, however, compensated by a special screening protocol. The selection of potential
abzymes was based on immunodetection of binders of the Schiff base
4 rather than of the immunizing hapten 5. Binders
of the aldimine were further screened for ,
-elimination of
-chloroalanine. This easily detectable reaction depends on the
deprotonation at C
which is an integral step in the by far largest
group of PLP-dependent reactions of amino acids (Scheme 1).
The synthesis of the haptens 5 and the
protein conjugates 6 (Fig. 1) was described previously (3,
11). The hapten
N-(5
-phosphopyridoxyl)-L-lysine
was coupled to maleylated carrier protein (12) which was keyhole limpet
hemocyanin or bovine serum albumin (BSA) for immunization and ELISA,
respectively. Monoclonal antibodies were generated as described
previously (11). The antibodies were purified by affinity
chromatography on protein G-Sepharose 4 Fast Flow from Pharmacia
Biotech Inc. The concentration of antibody was measured photometrically
(E280,mg/ml = 1.4).
The hybridoma supernatants
were screened by ELISA 10-14 days after the fusion. Maxisorp plates
from Nunc were coated with hapten-BSA conjugate 6 (10 µg/ml in 50 mM sodium carbonate, pH 9.6, 50 µl/well)
for 1 h at 37 °C, washed with washing buffer (phosphate-buffered saline, 0.05% Tween 20, 0.02% sodium azide), and
blocked with BSA (1% w/v in washing buffer, 300 µl/well) for 30 min
at 37 °C. The hybridoma supernatants (50 µl/well) were added and
the plates incubated for 1 h at 37 °C. The binding of the
antigen was detected, after a washing step, with an alkaline phosphatase-labeled second antibody from Sigma. Binders of
N-(5
-phosphopyridoxyl)-L-lysine
were selected by comparison of the binding of hapten-BSA conjugate,
maleylated BSA, and unmodified BSA.
The inhibitory effects of PLP (0.1-2 mM, depending on the antibody), PLP plus 25 mM D- or L-norleucine, and PLP plus 25 mM glycine on antibody-antigen binding were estimated. Diluted hybridoma supernatant or purified antibodies were preincubated with the inhibitors in bis-tris propane/NaCl (50 mM bis-tris propane, 140 mM NaCl), pH 7.5, at 37 °C for 30 min in the dark. The incubation mixtures were added into the wells that had been coated with antigen (see above), and the plate was incubated for 30 min at 37 °C in the dark. The amount of the bound antibody was measured after addition of an alkaline phosphatase-labeled second antibody from Sigma (for details, see Ref. 11).
Measurement of the Rate of Schiff Base FormationThe
purified antibodies and the D- or L-enantiomer
of N-acetyllysine were mixed in bis-tris propane/NaCl, pH
7.5, at 25 °C. The reaction was started by the addition of PLP. The
final concentrations of the antibody, PLP, and the amino acid were 2.5 µM, 16 µM and 1 mM,
respectively. The absorbance of the reaction mixture was monitored in
the range of 410-450 nm, depending on the antibody, with an HP 8453
spectrophotometer. This wavelength range corresponds to the absorption
band of the protonated Schiff base (2, 5).
The
production of pyruvate in the presence of 10 µM antibody,
100 µM PLP, and 10 mM D- or
L-enantiomers of -chloroalanine in bis-tris
propane/NaCl, pH 7.0, at 25 °C in the dark was measured with lactate
dehydrogenase and NADH. A control reaction without antibody was run
under the same conditions. Absorbance at 340 nm was measured with an
HP 8453 spectrophotometer. The calculation of all catalytic activities
is based on the concentration of binding sites of the antibodies.
The reaction mixtures (40 µl) contained 25 µM antibody, 1 mM PLP, 25 mM glycine, and 106 cpm of [2-3H]glycine (Isotopchim) with a specific radioactivity of 30 Ci/mmol in bis-tris propane/NaCl, pH 7.0. After an incubation of 1 h at 25 °C in the dark, the released tritium was measured in 10-µl samples as described previously (5).
Determination of Transaminase ActivityThe reaction
mixtures contained 5-10 µM antibody, 200 µM PLP, and 200 mM
D/L-alanine in bis-tris propane/NaCl, pH 7.5, at 25 °C in the dark. The increase in both absorbance at 325 nm ( = 8,300 M
1 cm
1; Ref. 13) and
fluorescence (excitation wavelength 325 nm, wavelength of maximum
emission 389 nm) was used to detect PMP as product of transamination.
The negative control without antibody was measured under the same
conditions. In the case of very low activity, detection of catalysis by
fluorescence is the method of choice because of its higher
sensitivity.
The reaction mixtures contained 25 µM antibody, 0.1-1 mM PLP (depending on the binding affinity of the individual antibodies for the cofactor as estimated by competition ELISA), and 100 mM amino acid in bis-tris propane/NaCl, pH 7.5, at 25 °C in the dark. Samples were taken during a period of 6 h, derivatized with Marfey reagent, and analyzed by reverse phase HPLC (14). Newly generated peaks were identified by comparison with reference substances.
Measurements of Dissociation Equilibrium ConstantsThe
Kd values of antibody 15A9
for the haptens 5 (Fig. 1), PLP, and PMP were determined by
measuring the quenching of the intrinsic fluorescence of the antibody
(excitation wavelength 280 nm; wavelength of maximum emission 342 nm).
The concentration of the abzyme was in the range of 0.01-0.8
µM. The measurements were performed at 25 °C in
bis-tris propane/NaCl, pH 7.5, with a Spex Fluorolog spectrofluorometer
and were corrected for the fluorescence of the ligand itself.
Kd
values were calculated
by nonlinear regression analysis.
Because the imine double bond
of Schiff base 4, which is essential for PLP to exert its
catalytic effect, was absent in the hapten
N-(5
-phosphopyridoxyl)-L-lysine
5 (Fig. 1), we introduced an additional screening step to
select from the 24 hapten-binding antibodies those that bind also the
planar aldimine 4. PLP readily forms Schiff bases with
primary amino groups in an uncatalyzed equilibrium reaction. Thus, a
competition ELISA of the antibody-antigen binding with PLP and amino
acids as inhibitors was used to identify the binders of the Schiff base
(Scheme 1). Binders of the aldimine were expected to be inhibited more
strongly by PLP plus amino acid than by PLP or the amino acid alone. As amino acid ligands for these competitive binding assays we chose D- and L-norleucine and glycine. Antibodies
13B10, 8H4, 15A9, 11C2, and 14G1 showed indeed that their binding to
the antigen was inhibited more strongly in the presence of PLP plus 25 mM glycine and PLP plus 25 mM D- or
L-norleucine than in the presence of PLP or the amino acid
alone (Fig. 2). The inhibition of antibody-antigen binding by the Schiff base 4 formed from PLP plus glycine indicates the existence of a binding site for the amino acid moiety of
the hapten, and the difference in inhibition by PLP plus norleucine and
PLP plus glycine reflects the contribution of the amino acid side chain
to the binding of the Schiff base. In antibodies 13B10, 15A9, and 11C2
the side chain of L-norleucine positively contributed to
the binding of the Schiff base. In antibody 14G1, a small positive contribution by the side chain of D-norleucine was
observed. In all cases, except for antibody 11C2, the contribution of
the side chain of the enantiomeric amino acid was negative. Antibody
8H4 bound the Schiff base with both D- and
L-norleucine less tightly than that with glycine. The
inhibition profiles of antibodies 5G12 and 6E9 are displayed in Fig. 2
to illustrate the binding properties of the great majority of the
antibodies which did not show a significant difference in the
inhibition by PLP and by PLP plus amino acids. Apparently, these
antibodies cannot accommodate the planar aldimine adduct in their
binding site. The inhibition tests were applied to supernatants as well
as purified antibodies. The observed differences were negligible.
The immunological test for aldimine binding was validated by the
detection of catalysis of aldimine formation by part of the selected
antibodies. Purified antibodies 13B10 and 15A9 catalyzed Schiff base
formation between PLP and N-acetyl-L-lysine,
and antibody 8H4 between PLP and
N
-acetyl-D-lysine. The stereospecificity of
directly measured aldimine formation thus correlates with the stereospecificity of aldimine binding as assessed by competition ELISA.
The best catalyst, antibody 15A9, showed a marked acceleration of the
condensation reaction at a PLP concentration of 16 µM, which is considerably below its K
d
value of 90 µM (Fig. 3). Under the same
conditions, antibodies 8H4 and 13B10 also showed significant rate
acceleration. Determination of the initial rate at higher concentrations of PLP and amino acid was not possible because the
formation of Schiff base would become too fast to be followed without
rapid-kinetics methodology. Antibodies 11C2, 14G1, 5G12, and 6E9 did
not catalyze aldimine formation.
Screening for Deprotonation at C
Deprotonation at C of
the substrate is an integral step in the reaction pathways of the by
far largest group of PLP-dependent reactions of amino acids
(Scheme 1). The substrate analog
-chloroalanine has been reported to
act as a mechanism-based inhibitor of several B6 enzymes
(15). Because of the good leaving group in the
-position, deprotonation at C
is spontaneously followed by
,
-elimination. The resulting aminoacrylate intermediate may react with protein side
chains or decompose to chloride, ammonia, and easily detectable pyruvate (Fig. 4). Pyruvate production may thus serve as
an indicator of C
-deprotonation. The potential catalysts,
i.e. the aldimine-binding antibodies 13B10, 8H4, 15A9, 11C2,
and 14G1, were screened for PLP-dependent activity toward
the D- and L-enantiomers of
-chloroalanine (see "Experimental Procedures"). Antibody 15A9 showed
,
-elimination activity toward
-chloro-D-alanine
(kcat
= 50 min
1), and antibody 13B10 proved active toward
-chloro-L-alanine kcat
= 5 min
1). Antibodies 8H4, 11C2, 14G1, 5G12, and 6E9 did not
show any detectable activity toward either enantiomer. The
N
-acetyl-L-lysine containing hapten
9 (20 µM) completely inhibited the
,
-elimination reaction of
-chloro-L-alanine
catalyzed by antibody 15A9, indicating that the catalytic effect of the
antibody is due to its specific binding sites.
Measurement of tritium release from [2-3H]glycine was
used to confirm the PLP-dependent antibody 15A9-catalyzed
deprotonation at C (for details, see "Experimental Procedures").
Three control experiments containing PLP plus glycine without protein,
PLP plus glycine and unspecific IgG, and antibody 15A9 plus glycine
without PLP, were performed. The amount of released tritium in the test sample was corrected for that in the control with PLP plus glycine and
corresponded to an estimated value of
kcat
7 min
1, considering an isotope effect of 10 and the
half-saturation of the antibody with the substrate glycine.
The specific absorption and fluorescence
properties make PMP an easily detectable product of the transamination
reaction. Antibodies 13B10, 8H4, 14G1, and 11C2 did not accelerate the
transamination reaction of PLP and D/L-alanine.
Antibody 15A9 was found to catalyze the transamination reaction of PLP
with D-alanine (Fig. 5) and other
hydrophobic D-amino acids (11). The
kcat value for
transamination with D-alanine was 0.42 min
1,
corresponding to a 5,000-fold rate acceleration compared with the
catalytic effect of PLP alone (11). Antibody 15A9 has been shown
previously by HPLC analysis not to catalyze any reaction of amino acids
other than transamination. The same analysis was applied to antibody
13B10 (see "Experimental Procedures"). No racemization,
decarboxylation, or elimination reaction with
D/L-alanine and
D/L-serine was observed.
Binding Affinity for Phosphopyridoxyl Amino Acids
The
dissociation equilibrium constants of antibody 15A9 for different
phosphopyridoxyl amino acids 5, PLP, and PMP were determined
by measuring the quenching of the intrinsic tryptophan fluorescence of
the antibodies upon addition of ligand (Fig. 6). A
comparison of the dissociation constants indicates the presence of
binding sites for both the cofactor and the amino acid moiety (Table
I). The antibody exhibits a relatively broad tolerance for the amino acid portion of the hapten. L-Amino acids as
well as D-amino acids can be bound. Remarkably,
3-amino-L-tyrosine is a very good ligand. The lowest
dissociation constant was measured with the hapten
N
-(5
-phosphopyridoxyl)-N
-acetyl-L-lysine,
which structurally resembles to a maximum extent antigen
6.
|
The multitude of possible transformations products of amino acids
(Scheme 1) is a major problem in the design of a screening procedure
for PLP-dependent catalytic antibodies. In view of this difficulty, we have devised a protocol that screens for the occurrence of two successive crucial reaction steps rather than for a final product. The first step for which the antibodies were screened was the
binding of the PLP-amino acid aldimine 4 (Fig. 1). This
selection, easily executed with a competition ELISA (Fig. 2), was
particularly important because we used, as in previous studies by other
laboratories (5-7), the reduced and thus nonplanar Schiff base
5 as hapten for the immunization. However, formation of the
extended planar resonance system, encompassing the pyridine ring of the
coenzyme and the imine double bond, is essential for the cleavage of
one of the bonds between C and its substituents (2). In the next
screening step, the antibodies were selected for catalysis of the
breaking of the C
-H bond, which in most PLP-dependent
reactions of amino acids follows the formation of the aldimine (Scheme
1). The substrate analog
-chloro-D/L-alanine provided a simple and generally applicable assay for deprotonation at
C
(Fig. 4). Only antibodies that catalyzed both aldimine binding and
-deprotonation were analyzed with HPLC for the generation of
specific reaction products from both enantiomers of different amino
acids.
The screening procedure clearly defines the requirements that have to
be met by an enzyme mimic to catalyze a PLP-dependent transformation of an amino acid (Scheme 1). As a corollary, the successive selection steps plausibly simulate the functional selection pressures that presumably were operative in the molecular evolution of
B6 enzymes. Comparison of amino acid sequences has shown
that the B6 enzymes are of multiple evolutionary origin
(16-18). As required for a PLP-dependent catalytic
antibody, the ancestor protein of a B6 enzyme family very
likely had to possess a PLP and an amino acid binding site with a
geometry that accommodated the planar aldimine. Competition ELISA (Fig.
2), as well as the Kd
values for haptens determined with antibody 15A9 (Table I), indicated
that both the coenzyme and substrate moiety interact with the
catalytically active antibodies. Recognition of the amino acid side
chain is evident from the enantiomeric specificity of the antibodies in
the competition ELISA (Fig. 2), which varies in kind and degree, as
well as from the order of preference of amino acids in aldimine
binding, which invariably was N-acetyl-lysine > norleucine > alanine > glycine.2 Although the immunizing hapten
was a derivative of an L-amino acid, two of the five
aldimine-binding antibodies preferred D-amino acids (Fig.
2). The type of binding of PLP to the antibodies has been examined only
in 15A9, the only antibody that catalyzes a transformation of an amino
acid other than
-chloroalanine. Antibody 15A9 appears to bind PLP
noncovalently (11). Both experiments with nonenzymic model
systems3 (19) and the residual activity of
mutant PLP-dependent enzymes without active-site lysine
residue (20, 21) have indicated that formation of the
coenzyme-substrate aldimine by transimination rather than de
novo formation from PLP and amino acid is not essential for
catalysis. The ubiquitous occurrence of the coenzyme-binding lysine
residue might reflect a historic trait rather than a mechanistic necessity (16, 22). Covalent binding of PLP probably was the very first
step in the molecular evolution of B6 enzymes. Primordial B6 enzymes presumably had to cope with low concentrations
of the cofactor. In contrast, at the high concentrations of PLP and
amino acid used in our experiments aldimine is preformed from unbound PLP and amino acid at a rate fast enough and present in a concentration high enough to serve directly as substrate for the abzymes.
The selection of aldimine-binding antibodies was followed by screening
for a catalytic effect, i.e. the cleavage of the C-H bond
of the substrate moiety. In the molecular evolution of B6 enzymes, the analogous step after acquiring the capacity of aldimine binding may be assumed to have been the development of a catalytic apparatus facilitating the cleavage of one of the bonds between C
and its substituents. The easily measured
,
-elimination of
-chloro-D/L-alanine (Fig. 4) served to test for
C
-deprotonation, which underlies the majority of
PLP-dependent reactions of amino acids (Scheme 1). Antibody
13B10 was found to catalyze the
,
-elimination of
-chloro-L-alanine which is consistent with its
enantiomeric binding specificity. In contrast, antibody 15A9, which
preferably binds the aldimine with L-amino acids (Fig. 2),
catalyzed both the
,
-elimination and the transamination reaction
exclusively with D-amino acids. Apparently, the C
-H bond
of the L-amino acid substrate is directed toward an inert
surface region of the antibody (11).
Three reactions were found to be catalyzed by the antibodies: formation
of aldimine, deprotonation at C as reflected by
,
-elimination of
-chloroalanine, and transamination. Catalysis of aldimine formation might reflect a favorable relative orientation of bound PLP
and amino acid.
,
-Elimination of
-chloroalanine and
transamination share one important feature: the crucial reaction steps
are proton transfers (Scheme 1). Apparently, in antibody 13B10 and 15A9
acid-base groups are positioned in proximity of C
and C
/C4
,
respectively. Alternatively, water molecules might have access to these
atoms and mediate the proton transfers. With antibody 15A9,
transamination is 2 orders of magnitude slower than
,
-elimination, suggesting that reprotonation at C4
is
rate-limiting.
Antibody 15A9 is the only antibody catalyzing the transformation of a
natural amino acid. The antibody is remarkably reaction-specific, transamination being the only reaction that is observed. The antibody accelerates the transamination reaction not only of PLP and an amino
acid but also in the reverse direction with PMP and an oxoacid as
substrates (11). The orientation of the C-substituents relative to
the plane of the resonance system of imine and coenzyme together with
the presence (and absence) of catalytically effective protein side
chains serving as general acid-base groups or modulating the electron
repartition in the coenzyme-substrate adduct are thought to determine
the reaction specificity in B6 enzymes (23, 24). In
contrast to the reaction specificity, the substrate specificity of 15A9
is less strictly defined, apparently all hydrophobic amino acids, and
oxoacids in the reverse reaction with PMP, are generally accepted as
substrates (11). Thus, the results of the successive steps in the
functional screening of PLP-dependent antibody catalysts
correspond to the molecular evolution of B6 enzymes also
with respect to the development of specificity. In the evolution of
B6 enzymes, specialization for reaction specificity clearly
preceded that for substrate specificity (16, 25). The analogy reflects
the interplay of chance and necessity being operative in both
cases.
We thank Beat Kunz, Hans Wendt, and Stefan Bienz for assistance in practical aspects of the work. We are grateful to Heinz Gehring and Hans Hengartner for helpful discussions.
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