(Received for publication, June 26, 1995; and in revised form, July 31, 1995)
From the
Chemical modification experiments have previously implicated
four amino acid residues in the mechanism of type I dehydroquinase from Escherichia coli. To further test their importance, these
residues were mutated, and the resulting mutants were expressed,
purified, and characterized. When the highly conserved, Schiff
base-forming lysine residue was mutated (K170A) the resulting enzyme
showed a 10
-fold reduction in catalytic activity, but
was still able to bind both substrate and product, as shown by a novel
fluorescence-based ligand-binding assay. This is consistent with
Lys-170 playing a central role in catalysis and shows that, although
forming a covalent bond with the substrate, it is not essential for
ground state binding of substrate or product. Conversely, substituting
leucine for the conserved, iodoacetate-reactive methionine residue
(M205L) had little effect on k
or K
. Diethylpyrocarbonate experiments had
previously implicated either His-143 or His-146 as the putative active
site general base. Substituting alanine for each shows that H146A
retains full catalytic activity while H143A shows a 10
-fold
loss of activity. As with the K170A mutant, H143A can bind ligand, and
in addition to the predicted role of this residue as the
proton-abstracting general base, our data suggest that it is also
involved in the formation and breakdown of Schiff base intermediates.
Isoelectric focusing, electrospray ionization mass spectrometry, and
fluorescence spectroscopy show that the H143A mutant preferentially
stabilizes the formation of the product Schiff base, and that this
results in burst kinetics reminiscent of p-nitrophenyl acetate
hydrolysis by chymotrypsin. The most striking illustration of this
stabilization is the fact that the H143A mutant is isolated from
overexpressing cells with a significant proportion of the enzyme
monomers covalently bound to the product, 3-dehydroshikimate, via a
Schiff base linkage. Our data suggest that the H143A mutant is able to
slowly transform substrate to product but that the hydrolytic release
of the product is stalled. The proposed dual role of His-143 in the
mechanism of type I dehydroquinase may explain why the elimination
reaction catalyzed by this enzyme proceeds with syn stereochemistry.
Dehydroquinase (DHQase; 3-dehydroquinate dehydratase) ()catalyses the dehydration of 3-dehydroquinic acid to
3-dehydroshikimic acid and is the third step in the central shikimate
pathway of microorganisms, fungi, and plants(1) . The end
products of the pathway are the precursors of the aromatic amino acids,
and so the enzymes involved in these biosynthetic reactions have been
studied as potential sites for antimicrobial agents and
herbicides(2) . The focus of the present work is the mechanism
of the dehydroquinase enzyme from Escherichia coli (EC
4.2.1.10). In particular, the roles of several amino acid residues have
been investigated which chemical modification experiments have
implicated as either being involved in the mechanism or as being in
close proximity to the active site.
There are two distinct classes
of DHQase enzymes, designated type I and type II, whose properties and
mechanisms have been compared previously (3) . Type II DHQases
are thermostable enzymes with a relatively small subunit molecular mass
(16 kDa) but which oligomerize to form dodecamers. They were
originally identified as part of a catabolic pathway for utilization of
quinic acid in fungi(4) , but have since been found in the
shikimate pathway of a number of
prokaryotes(5, 6, 7) . The stereochemistry of
dehydration for the type II enzyme of Anacystis nidulans has
been identified as anti(8) , and it is thought that
the elimination of water occurs through a concerted mechanism without
the involvement of covalent intermediates (3, 9) . The
type I class, typified by the E. coli and Salmonella typhi enzymes, are dimers of 25-kDa monomer molecular mass and are not
heat-stable(10, 11) . The E. coli enzyme in
particular has been studied extensively, and the current model for its
mechanism is shown in Fig. S1. Chemical modification and peptide
mapping studies have established that Lys-170 forms a Schiff base with
the substrate and product; the latter intermediate can be trapped by
reduction with sodium borohydride(12, 13) . It has
also been shown that reduction of this intermediate has profound
effects on the stability of the resulting
protein(14, 15) . Lys-170 is conserved in all the
known type I DHQase
sequences(11, 13, 16, 17) , which is
consistent with its central role in the mechanism. Stereochemical
experiments have shown that, unlike the reaction catalyzed by the type
II class, the reaction is a syn elimination(9, 18) . Since anti elimination is chemically the preferred course for the uncatalyzed
reaction(18) , it has been suggested that the formation of the
Schiff base involves some distortion of the carbocyclic ring of
dehydroquinate to render the pro-R proton more
reactive(9) .
Figure S1: Scheme 1Proposed mechanism for type I dehydroquinase. His-143 is thought to be the general base, B.
The pH dependence of V for E. coli DHQase reveals the presence of a single
ionizing species with a pK
of
6.2(10) , consistent with the action of a general base in the
mechanism (Fig. S1). The identity of this base has been inferred
from diethyl pyrocarbonate experiments in which the modification of a
single histidine residue led to inactivation of the enzyme, and the
pK
of this modification corresponded to the
pK
determined from the
pH-V
profiles of the enzyme(19) .
Peptide mapping of the modified enzyme and sequencing of an isolated
peptide established that the reactive histidine residue was either
His-143 or His-146, and on the basis of sequence comparisons it was
concluded that His-143 was the putative general base B highlighted in Fig. S1.
Iodoacetate acts as an affinity labeling reagent of E. coli DHQase, destroying activity by alkylating two unusually reactive methionine residues, Met-23 and Met-205(20, 21) . Of the two residues, only Met-205 is completely conserved in the type I family. Alkylation of both residues is required for complete inactivation of the enzyme, implying that these residues may not be crucial for enzyme activity but may lie close to the active site. Moreover, the carboxymethylation of Met-205 can be reversed by treatment with 2-mercaptoethanol but only when the enzyme is in the folded state(21) . This was interpreted by Kleanthous and Coggins (21) as demonstrating the close proximity of a neutralizing positive charge near the carboxylate moiety of the carboxymethylated Met-205.
At present, there are no
three-dimensional structures for either class of DHQase, but high
resolution data should become available in the near future, since
diffracting crystals of both a type I (22) and a type II (23) enzyme have been produced. In the absence of such data the
roles of all the amino acids thus far identified by chemical
modification need clarification. Toward this end we have mutated
residues His-143, His-146, and Lys-170 to alanine and Met-205 to
leucine and characterized the resulting mutant proteins to elucidate
further the roles of the wild type residues in the mechanism of type I
DHQase. The mutations addressed several as yet unanswered questions
concerning the roles of these residues. The histidine mutations were
made for two reasons, first to determine unambiguously which of the two
was the likely DEPC-reactive residue and second to assess the
properties of the essential histidine to alanine mutation in terms of
the known mechanism. Met-205 alone was mutated since it is the only one
of the two reactive methionine residues which is conserved in the type
I family and Met-23 has already been mutated and shown to have no
effect on enzyme activity. ()Alanine was substituted for the
Schiff base-forming lysine to assess the relative importance of this
residue in catalysis and ground state binding of ligand.
Figure 1:
Isoelectric focusing gels of wild type
and mutant dehydroquinases. A, lane 1, wild type
DHQase; lane 2, H143A DHQase as prepared; lane 3,
K170A DHQase as prepared; lane 4, M205L DHQase as prepared; lane 5, a hybrid mixture of wild type DHQase containing dimers
with either none, one, or two subunits labeled with covalently bound
dehydroshikimate, prepared as described previously(30) . B, treatments of H143A DHQase. Lane 1, protein as
prepared; lane 2, after unfolding in 6 M guanidine
hydrochloride followed by refolding during dialysis; lane 3,
after dialysis in 50 mM NHOH
HCl + 50
mM Tris, pH 7.0 (final); lane 4, after addition of
dehydroquinate (500 µM for 30 min); lane 5, wild
type enzyme after addition of dehydroquinate (500 µM for
30 min). C, lane 1, H146A DHQase as prepared; lane 2, H146A DHQase after inactivation with dehydroquinate
and NaBH
(according to Kleanthous et
al.(14) ); lane 3, wild type enzyme as prepared; lane 4, wild type enzyme after inactivation with
dehydroquinate and
NaBH
(14) .
The H143A mutant showed a surprisingly complex pattern on IEF gels (Fig. 1A, lane 2); as prepared, it consists of three distinguishable species ranging from pI 4.6 to 4.9. This pattern is reminiscent of that seen when the Schiff base intermediate in wild type enzyme is first reduced with sodium borohydride (to yield a modified dimer of pI 4.6) and mixed with unmodified enzyme under denaturing conditions, and the protein mixture is allowed to refold (30) . In addition to the recovery of modified and unmodified dimers this treatment also results in the formation of a hybrid dimer (pI 4.75) in which one subunit carries a covalently bound product molecule while the other remains unmodified. Such a hybrid mixture was run as a control in Fig. 1A (lane 5). A very similar pattern is observed for the H143A mutant as purified (Fig. 1A, lane 2). However, in contrast to the hybrid mixture derived from wild type enzyme which is fixed, the extent of modification in the H143A mutant can be altered in several ways. 1) Unfolding of the protein in 6 M guanidine hydrochloride, followed by dialysis to remove the denaturant, completely removes the modification to yield a protein with the same pI as wild type dimer (Fig. 1B, lane 2). It is known that refolding of the wild type, dimeric enzyme occurs under these conditions with 100% recovery of folded protein(30) . 2) Dialysis against pH 7.0 buffer containing 50 mM hydroxylamine also removes the modification and again results in a protein with the same pI as wild type (Fig. 1B, lane 3). 3) Incubation with substrate under equilibrium conditions increases the extent of modification such that only modified dimer is now apparent (Fig. 1B, lane 4). Wild type enzyme under similar conditions (in which the Schiff base is not reduced) simply behaves as unmodified enzyme (Fig. 1B, lane 5). H143A mutant which had been cleared of endogenously bound ligand by hydroxylamine dialysis or guanidine hydrochloride denaturation can also be remodified with substrate, indicating that the removal of the modification is not an irreversible consequence of these treatments, and completely modified protein can be returned to the unmodified state proving that the denaturants do not selectively destroy modified protein (data not shown). In all subsequent experiments on this mutant the heterogeneity was removed by prior dialysis against buffers containing hydroxylamine.
The H146A mutant was also analyzed by isoelectric focusing (Fig. 1C, lane 1). Unlike the H143A mutant, a single isoelectric variant was observed. However, this mutant is clearly more acidic than H143A (pI 4.6), a property that is distinctly different from all the other mutations generated in this study; these all behaved like the wild type enzyme on IEF. In addition, borohydride reduction of ligand was required to trap the product Schiff base (again, unlike H143A) which further reduced the pI of the protein to 4.3 (Fig. 1C, lane 2). It is interesting to note that of the three mutations, H143A, H146A, and K170A, which could potentially increase the acidity of DHQase by removing a positive charge, only the H146A substitution produced this effect. Although circumstantial, this suggests that His-143 and Lys-170 do not carry positive charges at the isoelectric point of the native enzyme.
The M205L mutant had a slightly increased K and decreased k
compared to wild type
enzyme (Table 1); k
/K
shows a decrease of
3.5-fold. These changes are small in
comparison with those seen for mutations of the other supposed active
site residues and indicate that while Met-205 may be close to the
active site, it does not play any significant role in the mechanism of
the enzyme.
The H146A mutant had K and k
values very similar to wild type enzyme (Table 1). Replacement of this amino acid with alanine clearly
has little effect on the enzyme, ruling it out both as the site of
diethyl pyrocarbonate modification and as a mechanistically important
residue.
Kinetic analysis of the H143A mutant required that it was
first ``cleared'' of endogenously bound ligand. This was
accomplished (as described above and under ``Materials and
Methods'') by a 24-h dialysis against hydroxylamine-containing
buffer followed by removal of the hydroxylamine by a further dialysis
step. As with the lysine mutant, homogeneous H143A was practically
inactive under normal assay conditions. However, unlike the K170A
mutant, when large quantities of enzyme (50-200 µg/ml;
2-8 µM) were used in the assay, an initial burst of
absorption at 234 nm was observed, which corresponded to the formation
of 0.3-0.5 equivalents of product (based on the extinction
coefficient of free 3-dehydroshikimate), followed by a very slow steady
state increase (Fig. 2). The initial phases of these traces
could be fitted to a single exponential, giving rates of 2.6
10
s
in each case. The steady
state rates changed with substrate concentration in a hyperbolic
fashion and, when they were fitted to the Michaelis-Menten equation,
gave a K
similar to the wild type enzyme, whereas k
was reduced by 5-6 orders of magnitude
relative to wild type (Table 1). It is possible that some of the
residual activity observed in these preparations could be due to
contamination with wild type enzyme. However, it seems very unlikely
that this could be responsible for the slow burst at 234 nm that is
observed with this mutant (wild type contamination would simply result
in instant steady state rates). Furthermore, both mass spectrometry and
fluorescence spectroscopy (see below) suggest that this slow burst
represents accumulation of the product 3-dehydroshikimate at the active
site of the H143A mutant.
Figure 2: Burst kinetics of H143A dehydroquinase observed by UV spectrophotometry at 234 nm. The cuvette contained 100 µM dehydroquinate and different amounts of H143A protein: A, 1.9 nmol; B, 3.8 nmol; C, 5.8 nmol; D, 7.7 nmol, in a final volume of 1 ml of 50 mM potassium phosphate buffer, pH 7.0.
The masses for all mutants constructed in this study were analyzed by ESI-MS. The masses for H146A (observed 27,400.8 ± 0.2, calculated 27,400.7 Da), M205L (observed 27,448.7 ± 0.8, calculated 27,448.6 Da) and K170A (observed 27,409.32 ± 0.6, calculated 27,409.5 Da) all showed single peaks which corresponded to their predicted masses. In addition, K170A was incapable of forming a borohydride-susceptible Schiff base in the presence of substrate or product (data not shown). The purified H143A mutant does not show a single peak but two, one at 27,400.7 ± 0.7 Da, which corresponds to the calculated mass of the protein (27,400.6 Da), and the other at 27,554.6 ± 2.6 Da (Fig. 3A). The mass difference between these two peaks is 154 Da, which corresponds to the mass of the Schiff base of the product, 3-dehydroshikimate. Hydroxylamine-treated or guanidine hydrochloride-denatured enzyme (Fig. 3B) shows only one peak, at 27,401.2 ± 0.3 Da. When the H143A mutant which had been cleared of endogenously bound ligand was treated with substrate, a peak at 27,554.5 ± 0.7 Da reappeared and increased while that of the unmodified peak diminished (Fig. 3C).
Figure 3:
ESI-MS of H143A dehydroquinase. A, protein as prepared; B, protein after dialysis
against 50 mM NHOH
HCl + 50 mM Tris, pH 7.0 (final); C, after addition of dehydroquinate
(500 µM for 30 min).
The resolution of the ESI-MS data collected on all the DHQase samples (<2 Da) was sufficient to distinguish between product- and substrate-bound enzyme (which differ in mass by 18 Da). Nevertheless, we further analyzed the mass of the product-linked form of the H143A mutant using the maximum entropy software supplied with the mass spectrometer. Here again, only the product form was identified (data not shown).
We found that the solvent used for these mass
spectrometric measurements (1:1:0.001 acetonitrile:water:formic acid)
resulted in the slow removal of the modifying group that produces the
peak at 27,555 Da, and so experiments were conducted in such a way that
samples were added to the mobile phase of the mass spectrometer
immediately before injection and using a high flow rate. Using this
approach, it was possible to monitor the time-dependent accumulation of
product on the H143A mutant following mixing with substrate (Fig. 4). These data could be fitted to a single exponential
curve with a rate constant of 2.2 10
s
. Mixing substrate with wild type enzyme also
shows the appearance of a Schiff base-bound subunit (in agreement with
the work of Shneier et al.(34) ). This intermediate
did not, however, accumulate with time but remained at a constant level
(
10% of the unmodified peak).
Figure 4:
Formation of Schiff based-linked product
followed by ESI-MS. Accumulation of the dehydroshikimate Schiff base
adduct at the active sites of wild type () and H143A (
)
DHQase following exposure to the substrate, dehydroquinate. In both
cases, a solution of enzyme (0.86 mg/ml; 34 µM) was
treated with 500 µM dehydroquinate. Aliquots (20 µl)
were taken out at approximately 2-min intervals, mixed with 80 µl
of 1:1:0.001 MeCN:H
O:HCOOH, and injected into the ESI-MS at
a flow rate of 30 µl/min. Mass spectra were acquired over
approximately 1 min. The proportion of modified protein was calculated
from the ion counts for the peaks at 27,401 and 27,555 Da, and in the
case of the H143A data, the resulting curve was fitted to a single
exponential.
In most cases, it was possible to study only the effect
of an equilibrium mixture of substrate and product (K = 15; see ``Materials and Methods'' for further
details) on the fluorescence properties of wild type and mutant
enzymes, and the values resulting from this analysis are shown in Table 1. Addition of such a mixture to wild type enzyme caused a
decrease in fluorescence intensity but no significant change in the
wavelength of maximum emission (325 nm), implying that there is no
major change in the environment of the tryptophan residue (Fig. 5A). A plot of the change in intensity versus ligand concentration had the shape of a simple ligand binding
curve from which a K
was derived (Table 1).
Figure 5:
Ligand binding to dehydroquinase monitored
by tryptophan fluorescence emission spectrometry. A,
fluorescence spectra of wild type DHQase, following excitation at 295
nm, in the absence (upper trace) and presence (lower
trace) of an equilibrium mixture of DHQ/DHS (see ``Materials
and Methods''). B, titration of the K170A DHQase mutant
(50 µg/ml; 2 µM) with dehydroquinate () and the
equilibrium mixture of DHQ/DHS (
) followed at an emission
wavelength of 330 nm.
For the K170A mutant the effects of substrate alone and the ligand mixture on protein fluorescence could be distinguished because substrate and product are not interconverted during the course of the experiment (Fig. 5B and Table 1). It is notable from these data that the product is less strongly bound than the substrate in this mutant. Although a residue which makes a covalent bond to the substrate has been removed in the K170A mutant, the binding affinity for the equilibrium ligand mixture is only marginally affected.
The H143A mutant again shows features distinct from the
other DHQase mutants. The substrate and the equilibrium ligand mixture
can bind to this enzyme and cause a fluorescence quench, but this
process was found to be time-dependent, and in both cases the decay
could be fitted satisfactorily to a single exponential (see Fig. 6, where the curve for substrate is shown). At saturating
concentrations of substrate (such as is shown in Fig. 6, see
below) the rate of decay was 2. 5 10
s
. The rate constant determined for the
substrate/product mixture was similar (data not shown). The overall
magnitude of the ligand-induced fluorescence quench for H143A was
equivalent to that of wild type enzyme and was found to be
concentration-dependent. However, experiments conducted under
equilibrium conditions (with substrate alone and with the ligand
mixture) indicated that the dissociation constant was very low, and
attempts to determine it required significantly lower protein
concentrations than those used for the other proteins in this study, at
which point spectrometer noise became a problem. It was therefore
possible to say only that the K
for substrate
alone (and the substrate/product equilibrium mixture) for this mutant
lies below 1 µM. The fact that the fluorescence quench
saturates at a given concentration of ligand indicates that it is
likely to be the result of an event following the rapid binding of
ligand.
Figure 6: Time-dependent changes in the intrinsic tryptophan fluorescence of H143A dehydroquinase caused by the addition of substrate. The change in fluorescence emission of H143A DHQase (50 µg/ml; 2 µM) at 330 nm (excitation, 295 nm) on addition of 500 µM dehydroquinate was observed as a function of time. The data have been fitted to a single exponential.
Experiments with substrate and product analogues have shown that the active site imposes relatively strict requirements on binding. Analogues lacking the carboxylate group or the 4-OH group are very poor substrates, although deletion of the 5-OH has less effect(35, 36) . The 1-OH is also rather sensitive to replacement by both large and small substituents, which may imply that the conformation of the ring, and especially its flexibility are important; this is consistent with the inference that the syn elimination results from distortion of the substrate, rendering the pro-R hydrogen the more acidic(35, 36) . Ligand binding to both wild type and the mutant DHQases generated in this study results in a quench of the intrinsic tryptophan fluorescence of the protein ( Fig. 5and Table 1), and this would be consistent with some form of conformational change at the active site of the enzyme. However, whether this alteration in structure represents the postulated conformational change resulting in distortion of the substrate carbocyclic ring or is simply a result of the close proximity to the single tryptophan residue of DHQase, will become clear only when crystal structures of bound and free DHQase become available.
The
mutation of Lys-170 to alanine almost totally abolishes activity. This
makes it clear that formation of the Schiff base is indispensable for
catalysis in this enzyme; if it were simply acting as a tether for the
substrate, one might expect some residual activity at high substrate
concentrations, but this is not seen. The ligand binding studies,
however, show that substantial binding interactions remain; the K for substrate/product equilibrium mixture is
only about 3-fold higher than the K
for wild type
enzyme, and a low K
could be measured for
substrate (Table 1). This implies that the lysine must exert most
of its influence on the catalytic steps of the enzyme rather than the
binding of substrate or product. It might be expected that the covalent
bond between substrate and enzyme would make a significant contribution
to ground state binding, yet it is clear that this is not the case;
Lys-170 plays a predominantly catalytic role in the mechanism.
One of the major effects of substituting alanine for histidine at
position 143, in the forward direction, is to dramatically slow down
the rate at which the enzyme can form product at the active site. The
ESI-MS data indicate that, in the presence of substrate, the product
Schiff base accumulates at the active site with a rate constant of 2.2
10
s
(Fig. 4). A
similar rate constant (2.6
10
s
) is obtained from the pre-steady state phase
of the activity assays for H143A (Fig. 2), suggesting that this
too represents the accumulation of the product at the active site. The
amplitude of this burst phase corresponds to <0.5 equivalents of
free product for each active site, but this may not represent the true
extent of binding, as the extinction coefficient of material bound to
the enzyme is unknown. The slow accumulation of the product Schiff base
at the active site is coincident with a time-dependent fluorescence
quench (Fig. 6) that has a first order rate constant (2.5
10
s
) almost identical to
the values obtained by ESI-MS and the activity assays.
All of the
above observations would be consistent with the postulated role of
His-143 being the proton-abstracting general base. However, the H143A
mutant has another property which suggests that in fact it may also be
involved in the breakdown (and possibly formation) of the Schiff base
intermediates. The hydrolytic release of the product Schiff base from
the active site of the mutant is dramatically affected, even more so
than the rate of formation of this intermediate. As discussed above,
the rate of product accumulation is 2.5
10
s
, and this is approximately 60,000-fold
slower than the turnover number for the wild type enzyme. However, the k
for the alanine mutant is at least 2 orders of
magnitude slower than this (Table 1), indicating that k
is governed by the hydrolysis of the product
Schiff base in H143A. The most striking illustration of this product
stabilization effect is the fact that the enzyme, as purified, is
substantially modified by the product Schiff base, as shown by the IEF
and ESI-MS data ( Fig. 1and Fig. 3). A further point to
make is that the presence of His-143 at the active site of DHQase
appears to destabilize ligand binding since substituting this residue
for alanine increases the affinity for both substrate and product.
These phenomena, accumulation of product followed by its very slow release, are strongly reminiscent of the classic experiments of Hartley and Kilby (38) on the burst kinetics of chymotrypsin when assayed with ester substrates. The rate-limiting step in the hydrolysis of esters by chymotrypsin is deacylation of the acyl-enzyme intermediate, and this is readily observed with poor substrates such as p-nitrophenyl acetate(38) . The burst kinetics of the H143A DHQase mutant are therefore consistent with the hydrolytic release of the product, 3-dehydroshikimate, being the rate-limiting step for this enzyme in the forward direction.
So what is the role of His-143 in the mechanism of type I DHQase? Our data are consistent with this residue having a wide ranging influence in the mechanism of the enzyme, involving the central steps of base-catalyzed elimination (Fig. S1) as well as the formation and breakdown of the Schiff base intermediates, depicted in Fig. S2. In this latter role, His-143 is proposed to act as a general acid in the formation of the imine intermediate and then a general base in its hydrolysis. An appealing aspect of this mechanism is that, following its participation in either formation or hydrolysis of the covalent intermediates, it remains appropriately placed and in appropriate ionization state, for its subsequent role in the central steps of the mechanism. In addition, a single base at the active site, involved in both C2 proton abstraction and Schiff base formation, may also explain why the type I DHQase reaction proceeds with syn stereochemistry.
Figure S2: Scheme 2Proposed mechanism for Schiff base formation and hydrolysis catalyzed by His-143 (B). The scheme would be valid from both the substrate and product directions except that when product was involved the ring would contain a double bond. Data for the H143A mutant show that product release is rate-limiting in the forward direction.
The idea of a dual function for His-143 in the type I DHQase mechanism has precedent in the literature of aspartate aminotransferase. The elegant studies of the K258A mutant of aspartate aminotransferase by Toney and Kirsch (39, 40) showed that exogenous amines could perform the 1,3-prototropic shift catalyzed by the active site lysine in the wild type enzyme, which is formally analogous to the elimination step of type I DHQase. Furthermore, exogenous amines also catalyzed hydrolysis of the Schiff base formed from aspartate and the pyridoxal cofactor at the active site of the mutant enzyme. Preliminary attempts to demonstrate this external amine catalysis with H143A DHQase have been unsuccessful (data not shown). If such effects are operating they are below the background rates we are currently able to measure.
In conclusion, using site-directed mutagenesis we have investigated the properties of mutant enzymes in order to probe the mechanism of type I DHQase from E. coli. Two of the mutations (H146A and M205L) show unambiguously that these residues play no role either in catalysis or ligand binding. Substitution of the active site Schiff base-forming lysine residue for alanine renders the enzyme virtually inactive but still capable of binding both substrate and product. The role of the putative active site general base His-143 would appear to be more complex than previously thought since substituting it for alanine increases the enzyme's affinity for both substrate and product, drastically slows down the rate of product accumulation in the forward direction, and stalls the hydrolytic release of product from the active site.