On the basis of a model of the complex in which l-aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of l-aspartate were pre
Trang 1of aspartate ammonia lyase from Bacillus sp YM55-1
Vinod Puthan Veetil1, Hans Raj1, Wim J Quax1, Dick B Janssen2and Gerrit J Poelarends1
1 Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands
2 Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands
Aspartate ammonia lyases (also referred to as
asparta-ses) are microbial enzymes that catalyze the reversible
deamination of l-aspartate (1) to yield fumarate (2)
and ammonia (3) (Scheme 1) These enzymes have
been purified and characterized from a number of Gram-positive and Gram-negative bacteria, including Escherichia coli, Hafnia alvei, Pseudomonas fluores-cens, Bacillus subtilis and Bacillus sp YM55-1 [1–11]
Keywords
aspartase; aspartate ammonia lyase;
Bacillus; deamination; enzyme mechanism
Correspondence
G J Poelarends, Department of
Pharmaceutical Biology, Groningen
Research Institute of Pharmacy, University
of Groningen, Antonius Deusinglaan 1, 9713
AV Groningen, The Netherlands
Fax: +31 50 3633000
Tel: +31 50 3633354
E-mail: g.j.poelarends@rug.nl
(Received 23 January 2009, revised 6 March
2009, accepted 20 March 2009)
doi:10.1111/j.1742-4658.2009.07015.x
Aspartate ammonia lyases (also referred to as aspartases) catalyze the revers-ible deamination of l-aspartate to yield fumarate and ammonia In the pro-posed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-aspartate to form an enzyme-stabilized enediolate intermediate Ketonization of this intermediate eliminates ammonia and yields the prod-uct, fumarate Although two crystal structures of aspartases have been deter-mined, details of the catalytic mechanism have not yet been elucidated In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally char-acterized aspartase (AspB) from Bacillus sp YM55-1 On the basis of a model of the complex in which l-aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of
l-aspartate were predicted to be Thr101, Asn142 and His188 This postulate
is supported by the mutagenesis studies: mutations at these positions resulted
in mutant enzymes with reduced activity and significant increases in the Km for l-aspartate Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pKa of approximately 7 and an acidic group with a pKa of approximately 10 are essential for catalysis His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these resi-dues are most likely involved in substrate binding and in stabilizing the enediolate intermediate Mutagenesis studies corroborate the essential role
of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive The substrate-binding model and kinetic anal-ysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the lyases in the aspartase⁄ fumarase superfamily
Abbreviations
Ap, ampicillin; AspA, aspartase from E coli; AspB, aspartase from Bacillus sp YM55-1; FumC, fumarase C from E coli; RCSB, Research Collaboratory for Structural Bioinformatics; PDB, Protein Data Bank.
Trang 2The best studied example is the aspartase (AspA) from
E coli, for which the crystal structure has been
eluci-dated [12] AspA functions as a homotetramer, where
each monomer consists of 478 amino acid residues,
and is allosterically activated by its substrate (1) and
Mg2+ ions, which are required for activity at alkaline
pH [3,13] The enzyme has a rather narrow substrate
specificity; it is specific for 1 and 2, and only
hydroxyl-amine can substitute for 3 as a substrate [14–16]
Stereochemical, kinetic isotope and pH-rate studies
indicate that AspA catalyzes an anti-elimination
reac-tion, where an active site base (with a pKa of
approxi-mately 6.5) abstracts the proton from C3 of 1 to form
an enzyme-stabilized enediolate intermediate (4;
Scheme 2) [5,6,16–18] The proposed enediolate
inter-mediate (i.e aci-carboxylate) can rearrange to
elimi-nate ammonia (3) and form the product, fumarate (2)
The rate determining step is the cleavage of the
carbon-nitrogen bond, which may be facilitated by a
general acid that protonates the leaving ammonia
group Additional support for the formation of an
enzyme-stabilized enediolate as an intermediate during
the deamination reaction is provided by inhibition
studies demonstrating that 3-nitro-2-aminopropionate,
when present in its resonance-stabilized nitronate
(aci-form) state, binds very tightly to AspA as a
transition state analog [19] On the basis of the crystal
structure of AspA and site-directed mutagenesis
studies, two positively charged residues (Arg29 and
Lys327) were proposed to bind the two carboxylate
groups of 1 (C4 and C1, respectively) [12,20,21] It was
further postulated that Ser143 functions as the general
acid catalyst [21]
On the basis of these studies, a picture of the
cata-lytic mechanism of AspA has emerged [16] However,
this is far from complete and major issues remain unresolved One issue concerns the identity of the gen-eral base catalyst that abstracts the C3 proton, and that of other essential catalytic and substrate-binding residues Another issue concerns whether substrate binding induces a conformational change that moves other residues into the active site, as might be expected for an enzyme that is allosterically activated by its sub-strate The crystal structure of AspA does not address these questions because it was solved in the absence of
a bound ligand [12] Attempts to obtain a crystal struc-ture of AspA (or any other aspartase) complexed with substrate, product or a competitive inhibitor have thus far proved unsuccessful
In our studies, we focus on aspartase (AspB) from the thermophilic bacterium Bacillus sp YM55-1 [10,11] This aspartase is of considerable biocatalytic interest because of its high activity and enantioselec-tivity, relative thermostability and lack of allosteric regulation by substrate or metal ions [10,11] It also efficiently catalyzes the reverse reaction (i.e the addition of ammonia to fumaric acid) Moreover, the broad nucleophile specificity of AspB enables the use
of a range of alternative nucleophiles, such as methyl-amine, hydroxylmethyl-amine, hydrazine and methoxylmethyl-amine,
in the conjugate addition reaction [22] These proper-ties indicate that AspB is a promising biocatalyst for the synthesis of enantiopure N-substituted aspartic acids
The crystal structure of AspB (also in the absence of
a bound ligand) was recently solved (Fig 1) and shows that the overall topology and active site architecture of AspB are similar to those observed in AspA and fuma-rase C (FumC) from E coli, confirming its member-ship in the aspartase⁄ fumarase superfamily of enzymes [23] Similar to AspA, AspB functions as a homotetr-amer, but now each subunit is composed of 468 amino acid residues [11,23] The functional tetramer contains four active sites, each harboring residues from three different subunits [23] A structural model of the com-plex in which l-aspartate was docked manually into the active site of AspB suggested interactions that might be responsible for the binding and activation
– O2C CO2
– O2C CO2
NH3
NH3 + +
Scheme 1 Reversible deamination of L -aspartate catalyzed by
aspartase.
O–
O
– O2C
NH3
H H B:
O–
O–
– O2C
NH3
O–
O
– O2C + NH3
Arg-29 Lys-327
Ser-143
Scheme 2 A schematic representation of the catalytic mechanism of AspA
Trang 3(i.e polarization of the C4 carboxylate group) of the
substrate [23] It is noteworthy that this approach of
manual docking of the substrate in the active site,
which may provide valuable insight into how residues
at the active site interact with the substrate, is
compli-cated for AspA because this enzyme is allosterically
activated by its substrate [13] Hence, it is reasonable
to expect some significant differences between the
active site structure of the apoenzyme and that of
the enzyme–substrate complex In the docking model,
the C1 carboxylate group of l-aspartate forms
hydro-gen-bonding interactions with the hydroxyl group of
Thr187, the amino group of Lys324 and the amide
group of Asn326, whereas the C4 carboxylate group of
the substrate forms hydrogen bonds with the hydroxyl
groups of Ser140 and Thr141 [23] According to the
model, the amino group of l-aspartate forms
hydro-gen-bonding interactions with the side chains of
Thr101, Asn142 and His188 [23] In the present study,
we performed site-directed mutagenesis experiments
on all of these residues to provide further insight into
the mechanism for the AspB-catalyzed deamination
reaction The results obtained also have implications
for our understanding of the catalytic mechanism of
AspA
Results
pH dependence of the kinetic parameters of AspB The pH dependences of kcatand kcat⁄ Kmfor the AspB-catalyzed deamination of l-aspartate (1; Scheme 1) were determined in 100 mm sodium phosphate buffer over the pH range 6.0–10.0 at 37C Both parameters show a bell-shaped dependence on pH, with limiting slopes of unity on either side of the pH maximum, indicating that both a basic group (ascending limb) and acidic group (descending limb) are important for catalysis The pH dependences of kcat and kcat⁄ Kmare given by Eqns (1,2):
kcatðpHÞ¼ ðkcatÞmax=ð1 þ ½Hþ=K1þ K2=½HþÞ ð1Þ
kcat=KmðpHÞ¼ ðkcat=KmÞmax=ð1þ½Hþ=K1þK2=½HþÞ ð2Þ where K1 is the ionization constant of the basic group and K2 is the ionization constant of the acidic group, with both being important for catalysis [24]
A nonlinear least-squares fit of the pH-dependence
of log (kcat⁄ Km), which follows the ionizations in the free enzyme and the free substrate, to the logarithmic form of Eqn (2) gives pKa values of pK1= 7.1 ± 0.1 and pK2= 9.8 ± 0.2 (Fig 2) A nonlinear least-squares fit of the pH-dependence of log (kcat), which follows the ionizations in the enzyme–substrate com-plex, to the logarithmic form of Eqn (1) yields pKa values of pK1= 6.2 ± 0.4 and pK2= 11.3 ± 2 (data not shown) Because data could not be collected at high enough pH values to clearly define the descending limb (i.e the extension of these studies beyond pH 10
is precluded by enzyme denaturation), the pKa value
Fig 1 A close-up of the active site of AspB [23] The roles of the
key active site residues (Thr101, Ser140, Thr141, Asn142, Thr187,
His188, Lys324 and Asn326) and their interactions are discussed in
the text Suffixes A, B and C indicate that the residues originate
from three different subunits Prepared using PYMOL [39].
Fig 2 pH-dependence of log kcat⁄ Km for the deamination of
L -aspartate (1) by wild-type AspB ( ) and the T141A (d) and H188A ( ) mutants The curves were generated by a nonlinear least-squares fit of the data to the logarithmic form of Eqn (2) Errors given in the text are standard deviations.
Trang 4for the descending limb may be somewhat less than
the calculated value
Construction and purification of the AspB
mutants
The residues selected for mutagenesis in the present
study were Thr101, Ser140, Thr141, Asn142, Thr187,
His188, Lys324 and Asn326 (Fig 1) Similar to
wild-type AspB, all mutant proteins were constructed as
His6-tagged fusion proteins, produced in E coli
TOP10, and purified to > 95% homogeneity (as
assessed by SDS⁄ PAGE) using a Ni-based immobilized
metal affinity chromatography procedure [22] This
purification procedure is highly specific for His6-tagged
proteins, eliminating the possibility that wild-type and
mutant AspB enzymes isolated from E coli TOP10 are
contaminated with native aspartase from this host A
previous study has shown that the recombinant
His6-tagged wild-type AspB has only slightly reduced
activ-ity compared to the native protein (without fusion tag)
[22] The yield of mutant protein from cell culture
varied in the range 8–20 mgÆL)1
Each AspB mutant was analyzed by nondenaturing
PAGE (data not shown) The mutant enzymes were
found to migrate comparably with the wild-type
enzyme, which suggests that the oligomeric association
of the mutants was still intact and that gross
confor-mational changes are unlikely to be present The
struc-tural integrity of some mutants was also assessed by
circular dichroism (CD) spectroscopy (see below)
Mutagenesis of Thr101, Asn142 and His188
On the basis of a previously reported structural model
of the complex in which l-aspartate (1) was docked
manually into the active site of AspB, the residues
responsible for binding the amino group of 1 are
predicted to be Thr101, Asn142 and His188 [23] To
investigate the importance of these residues to the
mechanism of AspB, eight single site-directed mutants
were constructed in which Thr101 was replaced with an
alanine or serine (T101A and T101S), Asn142 with an
alanine or glutamine (N142A and N142Q) and His188
with an alanine, glutamine, lysine or arginine (H188A,
H188Q, H188K and H188R) These mutations are
expected to completely remove the functional side chain
(e.g T101A) or to replace the side chain with another
one that has a similar functional group (e.g T101S)
The activities of the mutants were assayed using 1 as
the substrate It was found that replacement of His188
with a glutamine, lysine or arginine essentially
abol-ished enzymatic activity (Table 1) Under the
condi-tions of the kinetic assays, no activity could be detected for these mutants A conservative estimate of the sensitivity of the assay indicates an at least 106-fold decrease in kcat⁄ Km compared to that of wild-type AspB Substitution of His188 with an alanine, how-ever, resulted in an active enzyme with an approxi-mately 57-fold reduction in kcatand a 1.8-fold increase
in Km, which results in a approximately 100-fold decrease in kcat⁄ Km Hence, the major effect of this mutation is on the value of kcat
The mutation of Thr101 to an alanine has a large effect on the catalytic efficiency of AspB For the T101A mutant, a plot of various concentrations of 1 versus the initial rates measured at each concentration remained linear up to 1 m Hence, the T101A mutant could not be saturated Accordingly, only the kcat⁄ Km was determined, and this parameter is reduced approx-imately 7100-fold compared to that of wild-type AspB (Table 1) The mutation of Thr101 to another residue with an aliphatic hydroxyl group (serine), however, has a less drastic effect on kcat⁄ Km For the T101S mutant, which could also not be saturated with 1, the
kcat⁄ Km is reduced only approximately 80-fold Replacement of Asn142 with an alanine resulted in a mutant enzyme without detectable activity (Table 1), emphasizing the importance of this residue to the mechanism of AspB However, substitution of Asn142 with a glutamine, which also contains a terminal amide group, resulted in an active enzyme with an approxi-mately 3000-fold reduction in kcat⁄ Km
The T101A, N142A and H188A mutants were ana-lyzed by CD, and the spectra of these mutants were comparable to that of wild-type AspB, indicating that
Table 1 Kinetic parameters for the deamination of L -aspartate by wild-type AspB and the Thr101, Asn142 and His188 mutants Steady-state kinetic parameters were determined in 50 m M sodium phosphate buffer (pH 8.5) at 25 C Errors are standard deviations.
ND, not determined (a conservative estimate of the sensitivity of the assay indicates an at least 10 6 -fold decrease in kcat⁄ Km com-pared to that of wild-type AspB) In those cases where no activity was detected upon prolonged incubation with substrate, kcat values are given as < 0.001.
Enzyme kcat(s)1) Km(m M ) kcat⁄ Km ( M )1Æs)1)
Trang 5the mutations did not result in any major conforma-tional changes (Fig 3A)
Mutagenesis of Ser140 and Thr141
To investigate the importance of Ser140 and Thr141, which are the two residues implicated in binding the C4 carboxylate group of 1 [23], to the mechanism of AspB, seven single site-directed mutants were con-structed (S140A, S140R, S140K, T141A, T141V, T141R and T141K), as well as two double mutants (S140G⁄ T141G and S140K ⁄ T141K) Mutation to Gly, Ala and Val is expected to remove the functional group, whereas mutation to Arg or Lys may introduce
a new functional group It was found that replacement
of Ser140 and Thr141 with an arginine or lysine essen-tially abolished enzymatic activity (Table 2) The T141K mutant had a low amount of activity (approxi-mately 40 000-fold reduction in kcat⁄ Km), whereas the S140R, S140K and T141R mutants had no detectable activity The T141V mutant and the two double mutants (S140G⁄ T141G and S140K ⁄ T141K) also were completely inactive Substitution of Ser140 and Thr141
by an alanine, however, resulted in active enzymes For the T141A mutant, there is a 133-fold decrease in
kcat and an approximately nine-fold decrease in Km
A
B
C
Fig 3 Far-UV CD spectra of wild-type (Wt) and mutant enzymes (A) Superimposed spectra of wild-type AspB and the T101A, N142A and H188A mutants (B) Superimposed spectra of wild-type AspB and the S140A and T141A mutants (C) Superimposed spec-tra of wild-type AspB and the T187A, K324A and N326A mutants Spectra were measured in 10 m M NaH2PO4 buffer (pH 8.5) at a protein concentration of approximately 5 l M
Table 2 Kinetic parameters for the deamination of L -aspartate by wild-type AspB and the Ser140 and Thr141 mutants Steady-state kinetic parameters were determined in 50 m M sodium phosphate buffer (pH 8.5) at 25 C Errors are standard deviations ND, not determined.
Enzyme kcat(s)1) Km(m M ) kcat⁄ Km ( M )1Æs)1)
Trang 6using 1 As a result, the kcat⁄ Km is reduced 15-fold.
The major effect of this mutation is observed in kcat
For the S140A mutant, only the kcat⁄ Km could be
determined, and this parameter is reduced 27-fold
compared to that of wild-type AspB Because the
increase in Kmfor the S140A mutant is > 27-fold, the
kcat must be > 40 s)1 Hence, the major effect of this
mutation is likely observed in Km The CD spectra of
the S140A and T141A mutants revealed no significant
differences compared to that of the wild-type protein
(Fig 3B)
Mutagenesis of Thr187, Lys324 and Asn326
The substrate-binding model suggests that residues
Thr187, Lys324 and Asn326 are responsible for
bind-ing the C1 carboxylate group of 1 [23] To investigate
the importance of these residues for AspB activity, 10
single site-directed mutants were constructed in which
Thr187 was replaced by an alanine or serine (T187A
and T187S), Asn326 with an alanine or glutamine
(N326A and N326Q) and Lys324 with an alanine,
serine, valine, histidine, arginine or aspartic acid
(K324A, K324S, K324V, K324H, K324R and
K324D) It was found that replacement of Lys324
with a small (alanine), polar (serine), charged
(histi-dine, arginine and aspartate) or hydrophobic (valine)
residue resulted in mutant enzymes with no detectable
activity (Table 3), emphasizing the essential role of
Lys324 in the mechanism of AspB Prolonged
incuba-tion with 1 revealed a small amount of activity for
the K324R mutant, whereas all other mutants had no
detectable activity The activity of the K324R mutant
is too low to measure kinetic parameters Mutations
at positions Thr187 and Asn326 resulted in active enzymes Substitution of Thr187 with a serine resulted
in a mutant (T187S) with a surprisingly improved kcat (approximately five-fold) compared to that of wild-type AspB The Km value, however, increased signifi-cantly (approximately 11-fold) As a result, the
kcat⁄ Km is reduced approximately 2.3-fold For the T187A, N326A and N326Q mutants, only the kcat⁄ Km values could be determined, and these values are reduced 6280-fold, 22 500-fold and 168 750-fold, respectively, compared to the kcat⁄ Km of wild-type AspB
The T187A, K324A, and N326A mutants were also analyzed by CD and showed no detectable differences from the wild-type protein (Fig 3C)
pH dependence of the kinetic parameters of the T141A and H188A mutants
To determine whether Thr141 or His188 was responsi-ble for the ascending limb (i.e the basic group on the enzyme important for catalysis) or descending limb (i.e the acidic group on the enzyme important for catalysis) of the pH-rate profile of wild-type AspB, the
pH dependence of kcat⁄ Km for the T141A- and H188A-catalyzed deamination of 1 was determined over the pH range 6.0–10 Similar to wild-type AspB, the pH-rate profiles of the T141A and H188A mutants (comprising the only mutations at these positions that resulted in an active enzyme) are bell-shaped with slopes of unity (Fig 2) For the T141A mutant, pKa values of pK1= 6.8 ± 0.1 and pK2= 11.1 ± 0.5 were found For the H188A mutant, pKa values of
pK1= 7.4 ± 0.3 and pK2= 9.4 ± 0.3 were found
Competitive inhibition of AspB and the T141A and H188A mutants
It has previously been reported that 3-nitropropionate and d-malate are competitive inhibitors of the deami-nation activity of AspA with Ki values of 0.83 and 0.66 mm, respectively [14] These observations prompted us to examine whether these compounds are also competitive inhibitors of AspB Lineweaver–Burk reciprocal plots using three or four different inhibitor concentrations demonstrate that 3-nitropropionate and
d-malate are competitive inhibitors of the aspartase activity of AspB with Ki values of 2.0 ± 0.5 mm and
68 ± 12 mm, respectively These compounds are also competitive inhibitors of the aspartase activity of the T141A and H188A mutants For the T141A mutant,
Ki values of 0.5 ± 0.1 mm and 48 ± 12 mm were found for 3-nitropropionate and d-malate, respectively
Table 3 Kinetic parameters for the deamination of L -aspartate by
wild-type AspB and the Thr187, Lys324 and Asn326 mutants.
Steady-state kinetic parameters were determined in 50 m M sodium
phosphate buffer (pH 8.5) at 25 C Errors are standard deviations.
ND, not determined.
Enzyme kcat(s)1) Km(m M ) kcat⁄ Km ( M )1Æs)1)
Trang 7For the H188A mutant, Kivalues of 0.01 ± 0.001 mm
and 0.4 ± 0.02 mm were found for 3-nitropropionate
and d-malate, respectively (Fig 4) The results show
that replacing the histidine at position 188 with alanine results in a mutant with a surprisingly improved affin-ity for 3-nitropropionate (approximately 200-fold) and
d-malate (approximately 170-fold)
Discussion
On the basis of sequence analysis and crystallographic observations with AspA [Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) entry 1JSW], AspB (RCSB PDB entry 1J3U) and FumC (in complex with a substrate analog, cit-rate; RCSB PDB entry 1FUO) [12,25], Fujii et al [23] have made a structural model of the complex in which
l-aspartate (1; Scheme 3) was docked manually into the active site of AspB Guided by this substrate-bind-ing model, we have selected eight active site residues for mutagenesis to provide further insight into the mechanism of AspB In addition, we have performed pH-rate and inhibition studies to further illuminate the mechanistic role of certain residues The results obtained in these studies are interpreted and related to the mechanisms of AspA and other superfamily enzymes
We first determined the probable chemical mecha-nism of the AspB-catalyzed reaction and pKavalues of potential acid and base catalysts by pH-rate studies From the ascending limb of the pH-rate profile of AspB, a residue with a pKa of 7.1 ± 0.1, which must
be deprotonated for optimal activity, may be the gen-eral base catalyst involved in abstraction of the C3 proton of 1 From the descending limb, a residue with
a pKa of 9.8 ± 0.2, which must be protonated for optimal activity, may be the general acid catalyst
A
B
Fig 4 Lineweaver–Burk reciprocal plots showing the competitive
mode of inhibition of the H188A mutant by 3-nitropropionate (A)
and D -malate (B) The curves were generated by fitting the data by
nonlinear regression analysis using the equation for competitive
inhibition, yielding the Kivalues given in the text.
O
-O
NH3
H H B:
O–
O–
–
O 2 C
NH3 +
Lys-324 Asn-326
Thr-141
Th r-1 01
Asn 4 -2 H is-18 8
Ser-140 B:H
Thr-141 O H
Ser-140 O H HN
N His-188
Thr-101 OH
+
Asn-142
H2N O
Thr-187 Lys-324
Asn-326
O O O
H
NH3
NH2
O
2 + 3
Scheme 3 A schematic representation of the proposed catalytic mechanism of AspB.
Trang 8involved in protonation of the leaving amino group of
1 However, it is important to emphasize that it is
presently unknown whether the protonation state of
the leaving group in the AspB-catalyzed reaction is
that of ammonia or ammonium ion [6,23] If the
amino group is released as ammonia, a general acid
catalyst may not be required for the reaction The
descending limbs of the kcat⁄ Km versus pH and kcat
versus pH profiles would then reflect the deprotonation
of the amino group of the substrate (the pKa of the
amino group of 1 = 9.8) and active-site bound
inter-mediate (i.e the putative enediolate), respectively
Hence, the observed pH dependence of the kinetic
parameters for the AspB-catalyzed deamination of 1 is
most simply explained in terms of the ionization of a
single basic group at the active site and the ionization
of the amino group of the substrate (1) or
enzyme-bound intermediate (4) (Scheme 3)
The substrate binding model of AspB suggests that
His188 is one of the residues that interacts, via
hydro-gen-bonding, with the amino group of 1 [23]
Intrigu-ingly, this histidine residue is conserved in FumC and
other fumarase⁄ aspartase superfamily members (e.g
argininosuccinate lyase, d-crystallin and
adenylosucci-nate lyase) but is replaced by Gln191 in AspA [23]
Mutations of the conserved histidine residue severely
impair catalysis in the former enzymes [26–28] These
results, taken together with crystallographic
observa-tions, have led to several proposals for the catalytic
role of the histidine [29–31] These include roles for the
histidine as the general base catalyst that abstracts the
proton from C3 of the substrate, the general acid
cata-lyst that protonates the leaving C2 group, or the
criti-cal residue that activates an active site water molecule,
which then functions as the C3 proton abstracting base
[29–31]
To assess its role in catalysis, we have mutated
His188 in AspB Examination of the kinetic properties
of the H188A mutant, which is the only mutation at
this position that resulted in an active enzyme, shows
that the major effect of this mutation is on the value
of kcat No significant change was observed in the CD
spectrum of this mutant, demonstrating that the loss
of activity resulting from replacement of His188 by
alanine is not a consequence of the loss of structural
integrity of the enzyme Taken together, these
observa-tions suggest an important role for His188 in catalysis
However, the pH-rate profile of the H188A mutant
has the same overall shape as that of wild-type AspB,
with pKavalues of approximately 7.4 and 9.4,
suggest-ing that the protonation or deprotonation of His188 is
not responsible for the observed loss of activity on
either side of the pH optimum Therefore, we conclude
that His188 does not function as the typical general acid or general base catalyst in AspB, which is consis-tent with the absence of this residue in AspA
One potential explanation for the loss in activity of the H188A mutant is that His188 could position and
‘lock’ the amino group of 1 in a favorable orientation for deamination In this scenario, loss in activity of the H188A mutant is a result of the removal of optimal hydrogen-bonding interactions with the amino func-tionality, locating this group in an unfavorable posi-tion for the eliminaposi-tion reacposi-tion Support for this view
is provided by an analysis of the substrate specificity
of the H188A mutant in the reverse amine addition reaction In comparison to the wild-type AspB-cata-lyzed addition of methylamine to fumarate, the H188A mutant displays a two-fold increase in kcat and a higher Km for methylamine, suggesting that His188 is one of the residues that influences the nucleophile (i.e amine) specificity of AspB (V Puthan Veetil &
G J Poelarends, unpublished results) Extrapolation
of this observation to substrate binding supports the proposed role for His188 in binding the amino group
of 1 (Scheme 3) The corresponding glutamine residue
in AspA (Gln191) may participate in a similar hydro-gen-bonding interaction with the substrate Another notable effect of the H188A mutant is the 170–200-fold increase in the binding affinity for the competitive inhibitors 3-nitropropionate and d-malate This sug-gests that the decrease in catalysis for this mutant may
be the result of a combination of effects, including the suboptimal positioning of the amino group and the slower release of product
In the substrate binding model of AspB, two other residues (Thr101 and Asn142) are implicated in bind-ing the amino group of 1 [23] The correspondbind-ing res-idues (Thr100 and Asn141) in the FumC–citrate complex were found to assist His188 (corresponding
to His188 in AspB) in binding an active site water molecule [23,25] This geometry suggests that these three residues may be responsible for the positioning and activation of the nucleophilic water molecule in the FumC-catalyzed hydration reaction, as well as the positioning and protonation of the leaving hydroxyl group in the reverse dehydration reaction To exam-ine the role of Thr101 and Asn142 in AspB, we have mutated these two residues Complete removal of the functionality at position Thr101 by replacement with
an alanine leads to a significant increase in Km (> 66-fold) and a large decrease in catalytic efficiency (> 7000-fold) The same substitution at position Asn142 even results in a complete loss of activity Both these alanine mutants retain their overall struc-tural integrity, as assessed by CD spectroscopy These
Trang 9observations, coupled with the enhancement in
activ-ity observed with a serine (position Thr101) or
gluta-mine (position Asn142) mutation at these positions,
suggest that the most likely role for Thr101 and
Asn142 is to participate in a hydrogen-bonding
inter-action with the amino group of 1 (Scheme 3)
Simi-larly, the corresponding residues in AspA (Thr104
and Asn145), which are positionally conserved but
have slighty different side chain orientations [12,23],
may assist Gln191 in binding the amino group of the
substrate
In the substrate binding model of AspB, the
hydro-xyl groups of Ser140 and Thr141 are hydrogen bonded
to the C4 carboxylate group of 1 [23] This suggests
roles for the hydroxyl functional groups of Ser140 and
Thr141 in binding the substrate, and possible
interme-diates, in the reaction mechanism of AspB
Crystallo-graphic studies on FumC support this view [25] In the
structure of the FumC–citrate complex, Ser139 and
Ser140 (corresponding to Ser140 and Thr141 in AspB)
are hydrogen bonded to one of the carboxylate groups
of citrate [23,25] This geometry suggests the
impor-tance of these residues in binding one of the two
carboxylate groups of the substrate In AspB, Ser140
and Thr141 are located at the N-terminal end of
a-helix 6 in the positively charged environment created
by a dipole moment of this helix [23]
In the present study, Ser140 and Thr141 in AspB
were mutated to assess their role in catalysis The best
characterized mutants are the S140A and T141A
mutants, which have measurable activity and show no
significant differences in their CD spectra compared to
that of the wild-type AspB Examination of the kinetic
properties for the S140A mutant shows that there is a
small effect on kcat⁄ Km and a larger increase in Km,
suggesting that Ser140 plays a major role in binding
the C4 carboxylate group of 1 (Scheme 3) By contrast
to these observations, examination of the kinetic
prop-erties for the T141A mutant shows that the major
effect of replacing Thr141 by alanine is on the value of
kcat This suggests an important role for Thr141 in
catalysis Another notable effect of the Thr141
muta-tion is the increase in the binding affinity for the
sub-strate (assuming that the Kmreflects substrate binding)
and for the competitive inhibitors 3-nitropropionate
and d-malate
A major part of the loss in activity of the T141A
mutant is likely a result of the removal of optimal
hydrogen-bonding interactions with the proposed
eno-late anion intermediate formed at the C4 carbonyl
position of 1, making the abstraction of a proton
from the C3 position less favorable Whether the
intermediate is an enolate anion (i.e a
resonance-stabilized carbanion) derived by abstraction of the C3 proton from 1, or an enol that could be obtained by protonation of an enolate anion, remains unknown Presumably, Thr141, together with Ser140, polarizes the C4 carboxylate group and stabilizes the enediolate intermediate (4) formed upon abstraction of the C3 proton (Scheme 3) This proposed role for Thr141 in the mechanism of AspB shares similarity with the one proposed for Glu317 in mandelate racemase [32] This enzyme catalyzes the equilibration of the (R)- and (S)-enantiomers of mandelate On the basis of muta-genesis, crystallographic and kinetic isotope studies, Mitra et al [32] proposed that Glu317 in mandelate racemase functions as a general acid catalyst in the concerted general acid–general base catalyzed forma-tion of a stabilized enolic tautomer of mandelic acid
as a reaction intermediate Glu317 can function as a general acid catalyst because it is protonated when substrate binds at the active site and it is properly positioned for partial proton transfer to the carboxyl-ate group of mandelic acid to form a strong hydro-gen-bonded enolic intermediate However, it is clear from the pH-rate profile of the T141A mutant of AspB that Thr141 is not the acidic group responsible for the descending limb of the pH-rate profile of wild-type AspB Therefore, we conclude that the protonation state of Thr141 does not show up in the pH versus kcat⁄ Km profile of AspB and that the descending limb of this profile is caused by deproto-nation of either the substrate or an unknown acidic group on the enzyme important for catalysis The observation that electrophilic catalysis by Thr141 in AspB (i.e there is a 133-fold drop in the value of kcat for the T141A mutant) is not as important as that by Glu317 in mandelate racemase (i.e there is a 4500-fold drop in the value of kcat for the E317Q mutant) [32] could be explained, at least in part, by the pres-ence of the dipole moment of a-helix 6, which likely assists Thr141 in stabilizing the negative charge that develops on one of the C4 carboxylate oxygens upon proton abstraction [23]
A comparison of the crystal structures of AspA and AspB shows that Ser140 and Thr141 of AspB are posi-tionally conserved as Ser143 and Thr144 in AspA (although the side-chain orientations are slightly different) [23] Replacement of Ser143 with glycine or threonine caused a significant decrease in kcat(10- and 100-fold, respectively) and a three- to four-fold increase in Km using 1 [21] On the basis of these observations, Ser143 in AspA was proposed to func-tion as the general acid catalyst that protonates the leaving C2 amino group (Scheme 2) [21] In view of the crystallographic observations with FumC [25] and
Trang 10the substrate-binding model of AspB [23], and taken
together with the mutagenesis results of the present
study, it is doubtful that Ser143 functions as the
general acid catalyst in AspA However, in the absence
of a crystal structure of AspA in complex with
substrate (or a substrate analog), we can only speculate
that the mechanistic roles of Ser143 and Thr144 in
AspA share similarity with those proposed in the
pres-ent study for Ser140 and Thr141 in AspB (Scheme 3)
In the structure of the FumC–citrate complex, one
of the other carboxylate groups of citrate interacts
with the side chain of Lys324 (corresponding to
Lys327 in AspA and Lys324 in AspB) [23,25]
Previ-ous studies have shown that mutation of Lys327 in
AspA results in a six-fold increase in Km and a large
(> 300-fold) decrease in kcat [20] These observations
suggest a role for the lysine residue in binding one of
the two carboxylate groups of the substrate
Accord-ing to the substrate-bindAccord-ing model of AspB, Lys324
binds the C1 carboxylate group of 1 [23] The
stron-gest support for this orientation of substrate in the
active site comes from the observation that
substitu-tion of Lys327 in AspA with an asparagine changes
the substrate specificity of the enzyme and allows it to
process l-aspartate-a-amide [33] Our experiments
clearly show that Lys324 is an essential residue in the
AspB-catalyzed reaction because mutations at this
position result in a complete loss of activity
More-over, the mutant enzymes appear to retain an intact
overall structural integrity (as shown for the K324A
mutant by CD spectroscopy) It is therefore
reason-able to conclude that Lys324 is crucial for substrate
binding through an interaction with the C1
carboxyl-ate group (Scheme 3)
In the region surrounding Lys324 in the structure
of the FumC–citrate complex, there are two residues
(Thr187 and Asn326) that are well positioned to assist
Lys324 in binding one of the citrate carboxylate
groups [25] A comparison of this structure with those
of AspA and AspB shows that these residues are
posi-tionally conserved as Thr190 and Asn329 in AspA
and Thr187 and Asn326 in AspB [23] The results
obtained in the present study support a role for
Thr187 and Asn326 in assisting Lys324 to bind and
position the substrate (through interactions with the
C1 carboxylate group of 1) (Scheme 3) because
mutants of these two residues display reduced activity
with large effects on the Kmfor 1 Similar mechanistic
roles may be proposed for the corresponding residues
in AspA
In summary, the mutagenesis and pH-rate studies
performed in the present study support the
substrate-binding model and initial mechanism reported by
Fujii et al [23] (Scheme 3), although other mecha-nisms cannot be ruled out In this mechanism, an active site base (with a pKa of approximately 7.1) abstracts the proton from C3 of 1 to form an enedio-late intermediate (4), which is stabilized by both Ser140 and Thr141 The interaction of two hydroxyl functional groups with one carboxylate group is con-sistent with a dianionic aci-carboxylate intermediate The identity of the residue with the pKa of 7.1 is not known (It has been suggested previously [23] that Ser318 may function as the general base in AspB However, this residue is not located in the presumed active site of the enzyme To establish whether the loop containing Ser318 may undergo a large confor-mational change upon substrate binding, positioning Ser318 in the vicinity of C3 of the substrate, we have initiated studies that aim to solve the X-ray structure
of the enzyme–substrate complex.) In the next step, the enediolate intermediate collapses and eliminates ammonia (3) to form the fumarate (2) product Thr101, Asn142 and His188 could position and ‘lock’ the amino group in a favorable orientation for deami-nation, whereas Thr187, Lys324 and Asn326 bind the C1 carboxylate group of the substrate Strong support for this arrangement in AspB (Scheme 3) also comes from the interactions observed in the recently solved crystal structure of the fumarase⁄ aspartase superfam-ily enzyme adenylosuccinate lyase complexed with adenylosuccinate [31] In the structure of this com-plex, Thr122 and Ser123 interact with the d-carboxyl-ate group (on which the negative charge accumuld-carboxyl-ates
in the aci-carboxylate intermediate) of the succinyl moiety of adenylosuccinate, whereas Thr170, Lys301 and Asn303 interact with the c-carboxylate group The positional conservation of these carboxylate bind-ing residues (where Ser140, Thr141, Thr187, Lys324 and Asn326 in AspB are replaced by Thr122, Ser123, Thr170, Lys301 and Asn303 in adenylosuccinate lyase) suggests a similar mode of substrate binding for these two superfamily members
The positional conservation of seven out of eight active site residues also suggests a mechanism for AspA that largely parallels that proposed for AspB (Scheme 3) where the residues in AspB (Thr101, Ser140, Thr141, Asn142, Thr187, Lys324 and Asn326) are replaced by the corresponding ones in AspA (Thr104, Ser143, Thr144, Asn145, Thr190, Lys327 and Asn329) This proposed mechanism needs to be corroborated by future crystallographic studies The utility of 3-nitropro-prionate as a potent competitive inhibitor and potential crystallographic ligand for AspB could help to identify the additional features that are necessary for a fully active and specific aspartase