Characterization of site-directed mutants of residues R58, R59, D116, W340 and R372 in the active site of E. coli cystathionine β-lyase

Article abstract of DOI:10.1002/pro.308

Cystathionine β-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce L-homocysteine, pyruvate, and ammonia. A series of active-site mutants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the k_cat/K_m^L-Cth for the β-elimination reaction range from a reduction of only 3-fold for D116A and D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of these residues for the hydrolysis of L-Cth is: R372?R58 > W340 ≈ R59 > D116. Comparison of the kinetic parameters for L-Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues W340 and R372 interact with the α-carboxylate moiety. The increase in the pK_a of the acidic limb and decrease in the pK_a of the basic limb of the k_cat/K_m^L-Cth versus pH profiles of the R58K and R58A mutants, respectively, support a role for this residue in modulating the pK_a of an active-site residue. Published by Wiley-Blackwell.

Full text of DOI:10.1002/pro.308

Characterization of site-directed mutants
of residues R58, R59, D116, W340 and
R372 in the active site of E. coli
cystathionine b-lyase
Pratik H. Lodha, Allison F. Jaworski, and Susan M. Aitken*
Department of Biology, Carleton University, Ottawa K1S 5B6, Canada
Received 9 October 2009; Revised 1 December 2009; Accepted 2 December 2009
DOI: 10.1002/pro.308
Published online 15 December 2009 proteinscience.org
Abstract: Cystathionine b-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce
L-homocysteine, pyruvate, and ammonia. A series of active-site mutants of Escherichia coli CBL
(
eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in
catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the
LꢀCth
kcat/K
for the b-elimination reaction range from a reduction of only 3-fold for D116A and
m
D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of
these residues for the hydrolysis of L-Cth is: R372 >> R58 > W340 ꢁ R59 > D116. Comparison of
the kinetic parameters for L-Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates
that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues
W340 and R372 interact with the a-carboxylate moiety. The increase in the pK of the acidic limb
a
LꢀCth
and decrease in the pK of the basic limb of the kcat/K
versus pH profiles of the R58K and
a
m
R58A mutants, respectively, support a role for this residue in modulating the pK of an active-site
a
residue.
0
Keywords: cystathionine; pyridoxal 5 -phosphate; site-directed mutagenesis; transsulfuration;
aminoethoxyvinylglycine; enzyme kinetics
Introduction
Cystathionine b-lyase (CBL) catalyses the hy-
drolysis of L-cystathionine (L-Cth) to produce L-homo-
cysteine (L-Hcys), pyruvate, and ammonia. This
reaction is the second step in the transsulfuration
pathway, which converts L-cysteine (L-Cys) to L-
Hcys, the immediate precursor of L-methionine (Fig.
1). The two enzymes of the transsulfuration path-
way, cystathionine c-synthase (CGS) and CBL, are
The enzyme cystathionine b-lyase is a target for the
development of new antimicrobial compounds to
address the challenge of increasing antibiotic resist-
ance. The design of novel inhibitors of this enzyme
will be facilitated by the information presented in
this manuscript on the roles of specific amino acids
in the active site and their interaction with sub-
strates and inhibitors.
1
unique to plants and bacteria. These enzymes are
attractive targets for the development of novel anti-
microbial compounds, to address the growing chal-
lenge of antibiotic resistance by microbial pathogens,
because methionine is a precursor for protein biosyn-
thesis as well as thiamine biosynthesis, via the ubiq-
uitous methyl donor S-adenosylmethionine, thereby
Abbreviations: AVG, aminoethoxyvinylglycine; CBL, cystathio-
nine b-lyase; CGL, cystathionine c-lyase; CGS, cystathionine
0
c-synthase;
DTNB,
5,5 -dithio-bis-(2-nitrobenzoic
acid);
L-Cth, L-cystathionine; L-Hcys, L-homocysteine; IPTG, Isopropyl-
b-D-thiogalactopyranoside; Ni-NTA, Ni-nitrilo triacetic acid; PLP
0
pyridoxal 5 -phosphate.
2
providing a link to DNA replication. A detailed
*Correspondence to: Susan M. Aitken, Department of Biology,
study of the mechanism of slow-binding inhibition
Carleton University, Ottawa K1S 5B6, Canada.
E-mail: susan_aitken@carleton.ca
of Escherichia coli CBL (eCBL) by the b,c-
Published by Wiley-Blackwell. V
C
2009 The Protein Society
PROTEIN SCIENCE 2010 VOL 19:383—391 383

Figure 1. A: The enzymes and metabolites of the transsulfuration pathway. B: The structures of the L-Cth substrate and
2
,3
slow-binding inhibitors of eCBL.
unsaturated amino acid aminoethoxyvinylglycine
AVG) and the structure of the eCBL-AVG complex
Although AVG lacks the distal carboxylate group of
L-Cth, an interaction is observed between R58
and the trifluoromethyl moiety of the N-hydrazino-
carbonylmethyl-2-trifluoromethylbenzamide inhibi-
(
3
were reported by Clausen et al. This work provides
a solid foundation for investigation of the roles of
active-site residues and the design of inhibitors, as
exemplified by the novel, slow-binding inhibitors of
eCBL, with IC50 values in the lM and sub-lM
2
tor designed by Ejim et al. The side chains of R58
and R59, which approach the bound inhibitor in the
˚
eCBL-AVG complex to within 3.6 and 6.7 A, respec-
2
range, recently developed by Ejim et al.
tively, have both been proposed to interact with the
3
,4,6
The structures of eCBL, yeast cystathionine c-
lyase (yCGL), the second enzyme of the reverse
transsulfuration pathway of mammals and yeast,
and E. coli CGS (eCGS) are so similar that the
distal carboxylate group of the L-Cth substrate.
A
series of site-directed mutants of the eCBL active-
site residues R58, R59, D116, W340, and R372 was
constructed to probe the specific roles of these resi-
dues (Fig. 2). The observed reduction in the activity
of the R58A,K, W340F, and R372A,K,L site-directed
mutants and the differences in their kinetic parame-
ters for hydrolysis of L-Cth and inhibition by AVG
identify R58, W340, and R372 as residues interact-
ing with the distal and a-carboxylate groups of the
L-Cth substrate.
r.m.s. deviation in their least squares superposition
˚
is only ꢂ1.5 A between ꢂ350 C
a
atoms of the protein
4–6
backbone.
Therefore, these enzymes provide a
useful model system to investigate determinants of
specificity, as differences in their substrate and reac-
tion specificity are likely the result of differences in
4–6
the placement and mobility of active-site residues.
The active site of the homotetrameric eCBL
enzyme is situated at the subunit interface of the
catalytic dimer and is comprised of residues from
RESULTS
All of the site-directed mutants were soluble and
yields were between 15–41 mg/L, which is compara-
ble to the 56 mg/L reported for the wild-type
4
each subunit. The crystal structures of eCBL in
complex with AVG, N-hydrazinocarbonylmethyl-2-
nitrobenzamide, and N-hydrazinocarbonylmethyl-2-
trifluoromethylbenzamide provide valuable insight
into the active site and mechanism of this enzyme
7
enzyme. The data for the R58K, R372A, and R372L
mutants could not be fit to the Michaelis–Menten
equation as saturation kinetics were not observed
2
–4
(
Fig. 1).
The a-carboxylate group of AVG interacts
within the solubility limit of the L-Cth substrate.
3
LꢀCth
with the side chains of W340 and R372 (Fig. 2).
Therefore, with the assumption that Km
>> [L-
3
84 PROTEINSCIENCE.ORG
Active-Site Residues in Cystathionine b-Lyase

3
Figure 2. Observed contacts of AVG in the active-site of eCBL. The dotted lines represent putative hydrogen bond
˚
distances of ꢃ 3.3 A between heteroatoms. The image was constructed using ChemDraw and PDB entry 1CL2.
Cth], the Michaelis–Menten equation was modified
enzyme, while those of R58K are increased by 14
and 29-fold, respectively (Table II, Fig. 5). Mutation
of R58 results in a shift in the basic and the acidic
limbs of the specific activity versus pH profiles of
R58A and R58K, respectively. The pH optima of the
R58A and R58K mutants are correspondingly shifted
to ꢂ8.5–9 and 9–9.5, respectively, compared with
LꢀCth
to obtain kcat/Km
values, but not independent
LꢀCth
estimates of kcat and Km
, for these enzymes (Ta-
ble I). The specific activity versus pH profile of
eCBL, and all of the site-directed mutants investi-
gated, is bell-shaped. Upon reaction of eCBL with
AVG, the 424-nm peak of the internal aldimine form
of the PLP cofactor shifts to 341 nm and an isobestic
point at 386 nm is observed, as described by Clausen
8.5–9.5 for the wild-type enzyme (Fig. 3). Therefore,
LꢀCth
the effect of pH on the kcat/Km
of R58A and
3
et al.
R58K, as well as R372K, which has an 345-fold
LꢀCth
reduction in kcat/Km
at pH 8.5, but an unaltered
pH optimum, was determined (Fig. 4). The values of
The D116A,N and R59A,K mutants
The pH optima of eCBL-D116A and D116N are
unchanged and the IC50 and K values, for inhibition
i
LꢀCth
Table I. Kinetic parameters of eCBL and site-directed
mutants
by AVG, and the kcat and Km
values, for L-Cth
a
hydrolysis, of these mutants are within ꢂ2-fold and
-4-fold, respectively, of the wild-type enzyme
Tables I and II). Similarly, the kcat, IC50, and K val-
ꢀ1
LꢀCth
LꢀCth
cat/K
m
ꢀ1 ꢀ1
Enzyme
k
cat (s
)
K
(mM)
k
(M
s
)
3
m
5
(
i
eCBL
R58A
R58K
R59A
R59K
D116A
D116N
W340F
R372A
R372K
34.1 6 0.6
9.7 6 0.3
n.s.
45.8 6 0.4
36.7 6 0.8
41.9 6 0.7
37.9 6 0.4
79 6 1
0.18 6 0.01
5.1 6 0.3
n.s.
1.02 6 0.03
1.51 6 0.09
0.67 6 0.04
0.55 6 0.02
1.35 6 0.05
n.s.
(1.9 6 0.1) ꢄ 10
3
ues of R59A are within ꢂ2-fold of the wild-type
(1.92 6 0.05) ꢄ 10
b
LꢀCth
385 6 1
enzyme, the pH optima is unchanged and the K
m
4
(4.5 6 0.1) ꢄ 10
of this mutant is increased by only 5.7-fold. In con-
4
(2.4 6 0.1) ꢄ 10
trast, while the kcat of R59K is within ꢂ2-fold of
4
(6.2 6 0.3) ꢄ 10
4
wild-type eCBL and the pH profile is unchanged,
(6.9 6 0.2) ꢄ 10
LꢀCth
4
the K
, IC50 and K
i
values of R59K are increased
(5.9 6 0.2) ꢄ 10
m
b
n.s.
8.1 6 0.8
n.s.
0.09 6 0.04
550 6 20
0.23 6 0.03
by 8.4, 29, and 6.3-fold, respectively (Tables I and II;
Fig. 3).
15 6 2
n.s
b
R372L
a
Kinetic parameters reported are for hydrolysis of L-Cth.
Reaction conditions: 2 mM DTNB, 0.01–6.4 mM L-Cth and
0.068–6.6 lM wild-type or mutant eCBL, depending on the
The R58A and R58K mutants
LꢀCth
The kcat and Km
of R58A are decreased 3.5-fold
ꢅ
activity of the enzyme, in assay buffer at 25 C. The data
and increased 28-fold, respectively, and the kcat
/
LꢀCth
were fit to the Michaelis-Menten equation to obtain kcat
K
of R58K, which could not be saturated within
m
LꢀCth
LꢀCth
and K
and equation 1 to obtain kcat/Km
.
m
the solubility limit of L-Cth, is increased ꢂ500-fold
Table I). In contrast, the IC and Ki values of
b
LꢀCth
n.s. indicates that Km
the L-Cth, such that kcat/Km
regression.
exceeds the solubility limit of
(
LꢀCth
50
was determined via linear
R58A are within 3-fold of those of the wild-type
Lodha et al.
PROTEIN SCIENCE VOL 19:383—391 385

Table II. Kinetic parameters for inhibition of eCBL and site-directed mutants by AVG
a
b
ꢀ1 b
ꢀ1 c
(s )
Enzyme
IC50 (lM)
1.7 6 0.2
Ki (lM)
k
2
(s
)
k
1
-
4
eCBL
R58A
R58K
R59A
1.9 6 0.6
6 6 1
(5 6 1) ꢄ 10
263
333
64
481
50
108
92
17.5
-
-
-
3
3
3
1.74 6 0.08
23 6 1
(2.0 6 0.4) ꢄ 10
(3.5 6 0.3) ꢄ 10
(1.3 6 0.4) ꢄ 10
55 6 7
1.34 6 0.05
4.6 6 0.3
2.6 6 0.4
3.3 6 0.6
43 6 6
2.7 6 0.9
12 6 3
3.9 6 0.9
2.7 6 0.8
40 6 10
n.d.
-4
R59K
(6 6 1) ꢄ 10
-
-
4
4
D116A
D116N
W340F
R372A
R372K
R372L
(4.2 6 0.9) ꢄ 10
(2.6 6 0.8) ꢄ 10
-4
(7 6 2) ꢄ 10
n.d.
n.d.
-
3
3500 6 1400
n.d.
9100 6 400
n.d.
(2.30 6 0.02) ꢄ 10
0.25
n.d.
a
Reaction conditions for IC50 measurements: Enzyme (0.024–4.3 lM, depending on the activity
4
ꢅ
of the mutant) was incubated with 0.05–10 lM AVG in assay buffer at 25 C for 10 min. Activ-
ity was subsequently measured (n ¼ 4) at a L-Cth substrate concentration of 0.1 mM and the
data were fit to equation 3 to obtain the IC50 value for each enzyme.
b
i 2
Reaction conditions for measurement of K and k : Wild-type eCBL and site-directed mutants
were incubated with 1.5 mM L-Cth and 0.005–7.5 mM AVG in assay buffer and the progress of
the reactions was monitored for 30 min. The progress curves were fit to equation 5 to obtain
k
obs values, which were plotted versus inhibitor concentration and fit to equation 6 to obtain the
values of k and K
Values of the rate constant k
equation K /k
¼ k
2
i
.
c
1
, for the association of eCBL and AVG were calculated using the
i
2
1
.
pKa1 and pK , corresponding to the acidic and basic
The W340, R372A, R372L and R372K mutants
The kcat of W340F is within ꢂ2-fold of the wild-type
enzyme and the pH profile of this mutant is
a2
LꢀCth
limbs of the kcat/Km
versus pH profile, respec-
tively, are presented in Table III. The pKa1 values of
R58A and R372K and the pKa2 values of R58K and
R372K are similar to the wild-type enzyme, differing
by only 0.01–0.24 pH units beyond the experimental
error, confirming the trend observed in the specific
activity versus pH profiles of these enzymes. In con-
trast, the pKa1 of R58K is increased 0.66 pH units
and pKa2 of R58A is decreased by 0.67 pH units, a
change of approximately 0.5 pH units beyond the ex-
perimental error in both cases (Table III, Fig. 4).
LꢀCth
unchanged. In contrast, the Km
, for hydrolysis of
L-Cth, and the IC50 and K values, for inhibition of
i
W340F by AVG, are increased by 10, 25, and 21-fold,
respectively (Tables I and II, Fig. 5). The kcat and
LꢀCth
K
of R372K are decreased 4.2-fold and
m
LꢀCth
increased 83-fold, respectively, and the kcat/Km
of
R372A and R372L, which could not be saturated
within the solubility limit of L-Cth, are reduced by
6
5
2.1 ꢄ 10 and 8.3 ꢄ 10 -fold, respectively (Table I).
The optimum pH of ꢂ8–9.5 of the R372K mutant is
similar to that of the wild-type enzyme. The low ac-
tivity of R372A and R372L precluded the
Figure 3. The pH dependence of specific activity for the
hydrolysis of L-Cth catalyzed by eCBL (ꢄ) and the R59A
LꢀCth
ꢀ1 ꢀ1
s ) for
(
*) and R59K (l) mutants site-directed mutants. Inset:
Figure 4. The pH dependence of kcat/Km
the hydrolysis of L-Cth catalyzed by eCBL (*) and the
R58A (l) R58K (D) site-directed mutants. Inset: Comparison
(mM
Comparison of the pH dependence of the specific activity
of eCBL (ꢄ), R58A (*) and R58K (l). Reaction conditions:
MBP buffer (50 mM MOPS, 50 mM Bicine and 50 mM
proline), 20 lM PLP, 2 mM DTNB, 6.25 mM L-Cth and 68
nM eCBL, 0.96 lM R58A, 0.56 lM R58K, 58 nM R59A or
LꢀCth
ꢀ1 ꢀ1
of the pH dependence of kcat/Km
(mM
s ) for eCBL
(*) and the R372K (^) mutant. Reaction conditions: MBP
buffer (50 mM MOPS, 50 mM Bicine and 50 mM proline),
20 lM PLP, 2 mM DTNB, 0.01–6.25 mM L-Cth and 0.068–
4.5 lM enzyme. The data were fit to equation 2.
0.13 lM R59K. Measurements were performed in
quadruplicate, as represented by the error bars.
3
86 PROTEINSCIENCE.ORG
Active-Site Residues in Cystathionine b-Lyase

LꢀCth
Table III. Parameters determined from the kcat/Km
freedom of rotation about the Ca-Cb bond of the sub-
strate in the enzymes of the c-subfamily. For exam-
3
versus pH profiles of wild-type eCBL and the R58A,
a
R58K and R372K site-directed mutants
ple, methoxyvinylglycine (MVG) acts as either a sub-
strate or an irreversible inhibitor of PLP-dependent
enzymes, depending on the nature of the active site
Enzyme
pKa1
pKa2
eCBL
R58A
R58K
R372K
8.28 6 0.06
7.93 6 0.08
8.94 6 0.09
8.44 6 0.09
10.20 6 0.06
9.53 6 0.09
9.8 6 0.1
3
,11–13
(
Fig. 1).
Comparison of the reaction of eCBL
with L-Cth, MVG, and AVG demonstrates that the
differing ability of active-site residues to form hydro-
gen bonds, tethering the distal portion of these mole-
cules, has an important effect on the ability of the
enzyme to catalyze their transformation. The distal
methoxy group of MVG lacks the hydrogen bonding
ability of AVG and L-Cth (Fig. 1) and is predicted to
have a correspondingly greater degree of mobility
and rotational freedom within the active site. As a
9.8 6 0.1
a
Kinetic measurements for the eCBL-catalyzed hydrolysis
of L-Cth were carried out from pH 6.4–10.6 in MBP buffer
containing 0.01–6.25 mM L-Cth, 20 lM PLP, 2 mM DTNB
ꢅ
LꢀCth
versus
and 0.068–4.5 lM enzyme at 25 C. The kcat/Km
pH data were fitted to equation 2 to obtain the values for
pKa1 and pKa2
.
3
result, MVG is deaminated by eCBL. In contrast,
investigation of their activity as a function of pH as
well as the reliable determination of inhibition pa-
rameters for these enzymes. The increases of 2100
AVG is a slow-binding inhibitor of eCBL because the
distal amino moiety of AVG enables it to hydrogen
bond to the side chain hydroxyl group of Y111,
thereby adopting a conformation which results in
the slowly-reversible formation of a ketimine inter-
i
and 4800-fold in IC50 and K , respectively, observed
for R372K are the most drastic of the 10 mutants
investigated (Table II, Fig. 5). Values of the rate con-
3
mediate (Figs. 1 and 2). The L-Cth substrate of
1
stant k for association of eCBL and AVG, calculated
eCBL possesses both distal amino and carboxylate
groups (Fig. 1) and is efficiently hydrolyzed to L-ho-
mocysteine and iminopropionate, via a b-elimination
reaction, which does not include the ketimine inter-
mediate observed upon reaction of eCBL with AVG.
using the equation K ¼ k /k , are within 5-fold of
i
2
1
the wild-type enzyme, with the exception of the 15
and ꢂ1000-fold increases in this parameter observed
for the W340F and R372K mutants (Table II). Both
of these mutations modify residues observed to inter-
act with the a-carboxylate moiety of the inhibitor in
3
the eCBL-AVG complex.
Residue D116
The structure of the eCBL-AVG complex provides a
useful guide for the selection of residues to probe
DISCUSSION
3
their role in binding of the L-Cth substrate (Fig. 2).
The identity, position, and flexibility of active-site
residues are key determinants of both the substrate
and reaction specificity of enzymes. This is particu-
larly true of enzymes dependent on the catalytically
versatile PLP cofactor. For example, the effect of a
single conservative modification on reaction specific-
ity is demonstrated by the b-elimination activity, not
observed for the wild-type enzyme, of the T81A,
S82A, T85A, Q157A,E,H, Y158F, and S289A
mutants of yeast cystathionine b-synthase and the
increased racemase and b-decarboxylase activities of
the R292K mutant of E. coli aspartate aminotrans-
ferase (eAATase), the archetypical PLP-dependent
However, the double bond of the b,c-unsaturated
8
–10
enzyme.
The c-subfamily of fold-type I of PLP-
dependent enzymes, including eCBL, eCGS, and
yCGL, provides a useful model system for the inves-
tigation of substrate and reaction specificity, as the
overall structures and many active-site residues are
Figure 5. The dependence of enzyme activity on AVG
concentration for wild-type eCBL (*) and the R58K (l),
D116N (ꢄ) and W340 (~) site-directed mutants. Inset:
Comparison of the effect of AVG inhibitor concentration on
4
–6
the activity of eCBL (*) and the R372K (^) mutant.
conserved in these enzymes.
The characterization
Reaction conditions: The enzyme (32 nM eCBL, 4.3 lM
R58K, 24 nM D116N, 57 nM W340 and 1.9 lM R372K) was
of a series of site-directed mutants of five eCBL
active-site residues (R58, R59, D116, W340, and
R372) is the focus of the current study.
4
mixed with 0.05–10 lM AVG in 50 mM Tris, pH 8.5,
ꢅ
containing 20 lM PLP, and incubated at 25 C for 10 min.
Clausen et al. have suggested, based on the
structure of the eCBL-AVG complex, that the parti-
tioning of bound substrate, or reactive intermedi-
ates, underlying reaction specificity, is related to the
Activity was subsequently measured in quadruplicate, as
represented by the error bars, at a L-Cth substrate
concentration of 0.1 mM and the data was fit to equation 3
to obtain the IC50 value for each enzyme.
Lodha et al.
PROTEIN SCIENCE VOL 19:383—391 387

LꢀCth
of the alanine and
inhibitor is not present in L-Cth and AVG does not
possess the sulfur atom at the c-position or the dis-
tal carboxylate group of the substrate (Fig. 1). The
ꢂ2-fold greater B-factor of the distal portion of AVG,
compared to the Ca region, demonstrates the rela-
tive mobility of this part of the inhibitor in the
fold increases in the kcat/Km
leucine substitution mutants of R372 (Table I),
respectively, and the three order of magnitude
i
increases in the IC50 and K values for inhibition of
eCBL-R372K by AVG (Table II) demonstrate the im-
portance of this residue (Fig. 2). The additional
3
˚
active site. Therefore, the binding mode observed
hydrogen bonds, and ꢂ1 A greater length, of the
for the distal portion of AVG may not be representa-
native arginine residue at position R372 of eCBL is
3
tive of the eCBL substrate (Fig. 2). Residue D116 is
expected to contribute to the two and three order of
LꢀCth
conserved as an aspartate in bacterial CBL sequen-
ces and as an arginine residue in bacterial CGS and
eukaryotic CGL. An interaction between the corre-
sponding R106 of eCGS and its O-succinyl-L-homo-
serine (OSHS) substrate was observed in docking
magnitude lower Km
and K for AVG, respec-
i
tively, of the native enzyme, compared with the
R372K mutant. The order of magnitude difference
between these parameters is likely due to the distal
carboxylate moiety of the substrate, which is lacking
in AVG. The tyrosine, phenylalanine, alanine, and
lysine substitution mutants of the corresponding
R386 of eAATase also cause a reduction of at least
5
studies, before energy minimization. The side chain
˚
of D116 is 8.3 A from the distal amino group of the
inhibitor in the eCBL-AVG complex (Fig. 2). This
residue was mutated to alanine and asparagine to
investigate the possibility that a conformational
change in the active site, upon binding of L-Cth,
could bring D116 into contact with the distal portion
of the substrate. However, in agreement with the
three orders of magnitude in kcat/K , demonstrating
m
that replacement with lysine resulted in a similar
10,14,15
effect as other nonconservative replacements.
Despite the lack of sequence similarity between
AATase and CBL, the two enzymes share the com-
mon catalytic core of fold-type I PLP-dependent
structure of the eCBL-AVG complex, the 3-4-fold
LꢀCth
4
increases in the Km
and the ꢃ2-fold increases in
enzymes as well as several active-site features. The
the IC50 and K
i
values of the D116A,N mutants dem-
4-fold decrease in the kcat of CBL-R372K contrasts
onstrates that this residue is not involved in binding
AVG or the distal amino group of L-Cth (Tables I
with the 55-fold decrease in this parameter reported
1
0,16
for AATase-R386K.
Vacca et al. proposed that
3
and II).
the observed loss of activity of the R386K mutant
reflects differences in the binding orientation and
conformation of the substrates, such that they are
Residue W340
1
0
The side chain nitrogen of W340 forms a hydrogen
bond with the a-carboxylate group of both the tri-
fluoroalanine and AVG inhibitors, but does not
interact with the corresponding carbonyl group of
N-hydrazinocarbonylmethyl-2-trifluoromethylbenza-
not optimally positioned for catalysis. Therefore,
the 14-fold smaller decrease in the kcat of eCBL-
R372K likely reflects differences in the nature of the
reactions catalyzed by these enzymes, as the hydro-
lysis of L-Cth is a facile reaction that, in contrast
with the transamination of eAATase, does not
require the generation of a ketimine intermediate or
the binding of a second substrate.
2
–4
mide (Figs. 1 and 2).
Residue W340 is conserved
in c-proteobacterial CBL, but is replaced by a leu-
cine residue in CGL from fungi and higher eurkar-
LꢀCth
yotes. The 10-fold increase in Km
and 21-fold increases in IC50 and K
and the 25
Residues R58 and R59 have been alternatively
proposed to interact with the distal carboxylate moi-
ety of L-Cth by Messerschmidt et al. and Clausen et
i
for inhibition
of eCBL-W340F by AVG support the proposed role
4
,6
for this residue in binding to the a-carboxylate
groups of L-Cth and AVG (Tables I and II).
al., respectively.
eCGS (OSHS þ L-Cys ! L-Cth) and yCGL (L-Cth !
) are R48/R49 and
The corresponding residues of
3
L-Cys þ a-ketobutyrate þ NH
3
The role of arginine residues in the active-site
of eCBL
R52/S53, respectively. Based on docking studies in
the eCGS active site, the distal carboxylate group of
OSHS and the a-carboxylate of L-Cys are proposed
The three arginine residues in the eCBL active site
were mutated in this study with the goal of probing
the role of each in the binding of L-Cth. The a-car-
boxylate groups of AVG and trifluoroalanine, in the
active sites of the eCBL-inhibitor complexes, both
form a pair of hydrogen bonds to the side chain of
5
to interact with R48 and R49, respectively. Clausen
et al. observed that the salt bridge between E235
and R59 of eCBL is similar to that between eAATase
residues D15 and R292, which also interacts with
the distal carboxylate of anionic substrates, and pro-
posed that eCBL-R59 could alternatively form an ion
pair with E235 in the free enzyme and with the dis-
tal carboxylate group of L-Cth, upon substrate bind-
3
,4
˚
2
R372 (Fig. 2). The low B-factor (ꢂ15 A ) of the a-
carboxylate group of the inhibitor in the eCBL-AVG
complex is a result of the interaction with R372, as
well as with the backbone amide nitrogen of S339
4
,17
ing.
However, docking of L-Cth to the active site
3
and the side chain of W340. The 83-fold increase in
of eCBL has indicated that the distal carboxylate
LꢀCth
6
5
6
the K
of R372K, the 2.1 ꢄ 10 and 8.3 ꢄ 10 -
group of the substrate interacts with R58.
m
3
88 PROTEINSCIENCE.ORG
Active-Site Residues in Cystathionine b-Lyase

Comparison of the kinetic parameters obtained in
this study for hydrolysis of the L-Cth substrate and
inhibition by the AVG inhibitor, which lacks the dis-
tal carboxylate moiety of L-Cth (Fig. 1), provides
in the pH 8.5–9.5 range, which is pH optimum of
7
,21
eCBL (Fig. 3).
Clausen et al. have suggested that
conformational changes in the active site, resulting
from substrate binding, would be expected to
increase the hydrophobicity of the active-site envi-
insight into the roles of R58 and R59. While the
LꢀCth
4
K
is increased 5.7 and 8.4-fold by mutation of
ronment and weaken the R58-Y111 interaction.
m
R59 to alanine or lysine, a 28-fold increase in this
parameter is observed for R58A and R58K could not
be saturated within the solubility limit of L-Cth. In
a
This would result in an increase in the pK of Y111,
thereby facilitating a role for Y111 in proton transfer
from the a-amino group of the L-Cth substrate, dur-
ing transaldimination, to aminoacrylate to facilitate
the release of the iminopropionate product. Corre-
spondingly, a shift in the specific activity versus pH
profiles was observed only for the R58A and R58K
mutants (Fig. 3). Vacca et al. reported a similar nar-
rowing of the specific activity versus pH profile for
the corresponding R292K mutant of eAATase, but
not for R386K (eCBL-R372K), and proposed that the
change may be due to deprotonation of K292, as the
contrast, the K
R59A, and R59K by AVG, were increased by only
.2, 29, 1.4, and 6.3-fold, respectively (Table II). The
i
values for inhibition of R58A, R58K,
3
similar changes, of less than one order of magnitude,
LꢀCth
in the K
i
and K values of R59A and R59K dem-
m
onstrate that this amino acid is not directly involved
in binding L-Cth and AVG. The observed minor
effects on the kinetic parameters of these mutants
are likely due to loss of the R59-E235 interaction
and the resulting alteration in active-site conforma-
tion. In contrast, the effect of the alanine and lysine
substitutions of R58 on the hydrolysis of L-Cth is
much greater than their effect on inhibition by AVG,
demonstrating that it is R58 that interacts with the
distal carboxylate of the substrate. Residue R58 also
interacts with both the phosphate group of the PLP
cofactor and the side chain hydroxyl of Y111 in the
10
a
pK of lysine is lower than that of arginine. How-
ever, a similar mechanism is unlikely in the case of
the eCBL-R58K mutant, unless the interaction of
R58 with the phosphate moiety of the PLP cofactor,
is weakened or not formed in the R58K enzyme (Fig.
LꢀCth
2). The kcat/Km
versus pH profiles of R58A and
R58K are also both narrower than that of the wild-
type enzyme. The value of pKa2, of the basic limb, of
4
eCBL active site (Fig. 2). In the structure of the
R58A is decreased by 0.66 pH units and pK , of the
a1
eAATase-methylaspartate complex the corresponding
R292 forms two hydrogen bonds each with the side
chain of D15 and the distal carboxylate group of the
substrate analog. Tethering of the eCBL-R58 side
chain by the phosphate group of PLP, similar to the
R292-D15 interaction of eAATase, may provide rigid-
ity to eCBL-R58 to enable the precise positioning of
the distal portion of substrate. The role of arginine
residues in the binding of anionic substrates, partic-
ularly carboxylate groups, has been observed in a
acidic limb, of R58K is increased by 0.67 pH units
a
(Fig. 4, Table III). The pK of the basic limb of the
LꢀCth
kcat/K
versus pH profile of eCBL may corre-
m
spond to the substrate, as it is within 0.6 pH units
of the pK of 9.63 determined for one of the amino
a
22
groups of L-Cth. The observed increase in the pKa
of the acidic limb of the R58K mutant may be due to
the decreased ability of a lysine in this position to
modulate the pK of Y111 to the same extent as R58
a
in the wild-type enzyme, as the e-amino group of
this residue cannot fulfill all of the hydrogen bond-
ing interactions observed (phosphate moiety of the
cofactor and side chain of Y111) and proposed (distal
carboxylate group of L-Cth) for residue R58. How-
ever, the increase in pKa1 and decrease pKa2
observed for R58K and R58A, respectively, are dis-
tinct to the each mutant, suggesting that the
10,17–20
wide range of enzymes.
˚
As a result of the ꢂ1-
A difference in the length of the lysine side chain
and its inability to fulfill the number of hydrogen-
bonding interactions formed by arginine, the e-
amino group of the R58K side chain cannot interact
with both the phosphate moiety of the cofactor and
the distal carboxylate group of the substrate in the
same manner as R58. The 29-fold increase in the Ki
for inhibition of R58K by AVG, which lacks a distal
carboxylate group, may reflect a weakening in the
interaction between Y111 and the distal amino group
of AVG in the context of the R58K mutation.
observed pK shifts may be an indirect effect result-
a
ing from a change in the conformation or charge dis-
tribution of the active site.
The CBL enzyme, which is unique to plants and
bacteria, is an attractive target for the development
of novel antimicrobial compounds because it is
linked to a variety of cellular processes, including
DNA replication, via the ubiquitous methyl donor S-
adenosylmethionine. This study has identified R58,
W340, and R372 as residues interacting with the
distal and a-carboxylate groups, respectively, of
the L-Cth substrate, information that will guide the
design of inhibitors of this enzyme. For example, the
addition of a distal carboxylate moiety to AVG would
Residue R58 may also be involved in modulating
the nucleophilic character of an active-site residue,
such as Y111, which forms a p-stacking interaction
with the pyridine ring of the cofactor and has been
proposed to abstract a proton from the a-amino
group of L-Cth and donate a proton to aminoacry-
4
late. The observed interaction between R58 and
Y111 (Fig. 2) may lower the pKa of the latter, such
that the side chain of Y111 exists as a phenolate ion
Lodha et al.
PROTEIN SCIENCE VOL 19:383—391 389

allow this compound to form a pair of hydrogen
bonds with residue R58 in the active site of eCBL.
in a three-component buffer, comprised of 50 mM
MOPS (pK ¼ 8.3) and 50
¼ 7.2), 50 mM Bicine (pK
The kinetic measure-
a
a
25,26
mM proline (pK ¼ 10.7).
a
Materials and Methods
ments were carried out at pH 6.4–10.6 in the pres-
ence of 20 lM PLP, 2 mM DTNB, 0.06–3.0 lM eCBL
Reagents
(
depending on the activity of the site-directed mu-
L-Cth and L-aminoethoxyvinylglycine (AVG) were
purchased from Sigma. Protease inhibitor (Complete,
EDTA-free) tablets were obtained from Roche. Ni-
tant) and 6.25 mM L-Cth, for specific activity meas-
urements, or 0.01–6.25 mM L-Cth, for the determi-
LꢀCth
nation of kcat/Km
. Specific activity versus pH
0
NTA resin was a Qiagen product. 5,5 -Dithiobis(2-
measurements were performed in quadruplicate to
provide an estimate of the experimental error associ-
nitrobenzoic acid) (DTNB) was from Pierce. Oligonu-
cleotide primers were synthesized by Integrated
DNA Technologies and mutants were sequenced by
DNA Landmarks before expression and purification.
ated with each point in the pH profile. The kcat
/
LꢀCth
K
versus pH data were fit to the bell-shaped
m
curve described by Eq. (2), where kcat/K mm ax is the
LꢀCth
24
upper limit for kcat/Km
at the pH optimum.
Construction, expression and purification of
site-directed mutants
kcat=K mm ax
þ 10pKa1ꢀpH þ 10pHꢀpKa2
kcat=Km ¼
(2)
Site-directed mutants were constructed by overlap-
extension polymerase chain reaction and inserted
into the pTrc-99aAF plasmid, which encodes an N-
1
7
terminal, 6-His tag and linker. Farsi et al. demon-
Inhibition of wild-type and site-directed mutants
of eCBL by AVG
strated that the presence of this affinity tag does not
7
alter the kinetic properties of eCBL. The E. coli
The IC50 values for inhibition of the eCBL enzymes
by AVG were determined by measuring enzyme
activity between 0.05 lM and 10 mM AVG. The
KS1000 metC:cat strain, in which the gene encoding
eCBL is replaced by that of chloramphenicol acetyl-
transferase, was employed for expression of the site-
directed mutants to avoid contamination with the
enzyme and inhibitor were mixed and incubated at
7
ꢅ
2
5 C for 10 min in assay buffer. Activity was subse-
wild-type E. coli enzyme. The wild-type and site-
quently measured at a L-Cth substrate concentration
directed mutants of eCBL were expressed and puri-
2
7
of 0.1 mM and the data were fit to Eq. (3). Meas-
fied as described by Farsi et al.
urements were performed in quadruplicate for each
enzyme. The parameters Actmax, Actmin, S and IC50
of Eq. (3) correspond to the maximal enzyme activ-
ity, the minimal enzyme activity, the slope of the
transition between the maximal and minimal activ-
ity plateaus and the midpoint of the transition,
respectively.
Determination of steady-state kinetic
parameters
Enzyme activity was measured in a total volume of
ꢅ
1
00 lL at 25 C using a Spectramax 340 microtiter
plate spectrophotometer (Molecular Devices). The
assay buffer was comprised of 50 mM Tris, pH 8.5,
containing 20 lM PLP, and the hydrolysis of L-Cth
Actmax ꢀ Actmin
0.01–6.25 mM) was detected (e412 ¼ 13,600 Mꢀ
1 ꢀ1
s
)
Act ¼
þ Actmin
(3)
(
ꢀ
ꢁ
S
0
I
via the reaction of 5,5 -dithiobis-(2-nitrobenzoic acid)
1 þ
IC50
7
,23,24
(
DTNB) with the free thiol of the L-Hcys product.
A background reading was recorded before initiation
of the reaction by the addition of eCBL (0.068–6.6 lM,
depending on the activity of the particular site-
Values for the dissociation constant K
rate constant k
i
and the
2
for the inhibition of eCBL by AVG
3
were determined as described by Clausen et al. In
directed mutant) for all assays. Values of kcat and
this model the enzyme and inhibitor do not form an
LꢀCth
K
for the hydrolysis of L-Cth were obtained by fit-
m
initial, rapidly reversible enzyme-inhibitor complex
3
ting of the data to the Michaelis-Menten equation and
[
Eq. (4)].
LꢀCth
kcat/K
was obtained independently from Eq. (1).
m
Data were fit by nonlinear regression with the SAS
software package (SAS Institute, Cary, NC).
k1½Iꢆ ðslowÞ
E
ꢀ
EI
(4)
k2
v
kcat=Km ꢄ ½Sꢆ
1 þ ½Sꢆ=Km
The wild-type eCBL and site-directed mutant
enzymes were incubated with 1.5 mM L-Cth and
.005–7.5 mM AVG in assay buffer and the progress
of the reactions was monitored for 30 min. Values of
obs were determined from the fit of Eq. (5) to the
progress curves. The resulting kobs values were plot-
ted versus inhibitor concentration and values of k
¼
(1)
½
Eꢆ
0
Evaluation of the pH dependence of wild-type
and site-directed mutants of eCBL
k
The pH dependence of L-Cth hydrolysis by eCBL
was determined using the continuous DTNB assay
2
3
90 PROTEINSCIENCE.ORG
Active-Site Residues in Cystathionine b-Lyase

and K
6).
i
were obtained from fitting of the data to Eq.
9. Quazi F, Aitken SM (2009) Characterization of the
S289A,D mutants of yeast cystathionine b-synthase.
Biochim Biophys Acta 1794:892–899.
(
Â
Ã
10. Vacca RA, Giannattasio S, Graber R, Sandmeier E,
Marra E, Christen P (1997) Active-site Arg-Lys substi-
tutions alter reaction and substrate specificity of aspar-
tate aminotransferase. J Biol Chem 272:21932–21937.
11. Rando RR (1974) b,c unsaturated amino acids as irre-
versible enzyme inhibitors. Nature 250:586–587.
ꢀ
kobst
ðvo ꢀ vsÞ 1 ꢀ exp
½
Pꢆ ¼ vst þ
(5)
(6)
kobs
k2½Iꢆ
kobs ¼ k2 þ
Kið1 þ ½sꢆ=KmÞ
1
1
1
1
1
1
2. Rando RR, Relyea N, Chong L (1976) Mechanism of
irreversible inhibition of aspartate-aminotransferase
by bacterial toxin L-2-amino-4-methoxy-trans-3-bute-
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acids to saturated a-keto acids by tryptophan synthase.
Biochem Biophy Res Commun 66:94–102.
Acknowledgments
The authors thank Heidi Los and Navya Kalidindi for
their technical assistance in protein purification and
the reviewers of this paper for their insightful com-
ments and suggestions. This work was supported by a
grant from the Natural Sciences and Engineering
Research Council of Canada.
4. Danishefsky AT, Onuffer JJ, Petsko GA, Ringe
D
(
1991) Activity and structure of the active-site mutants
R386Y and R386F of Escherichia coli aspartate-amino-
transferase. Biochemistry 30:1980–1985.
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Berger P, Gehring H, Jansonius JN, Christen P (1995)
0
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