A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity

Article abstract of DOI:10.1016/j.bbapap.2013.11.012

Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4-1.2 pH units, likely due to repositioning of the cofactor and modification of the pK_a of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3-3, 26-58 and 124-568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The K_m^l-Cth of E333 substitution variants is increased ~ 17-fold, while K_m ^l-OAS is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (k _cat/K_m^l-OSHS = 7 ± 2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (k_cat/K_m^l-Cth = 2100 ± 100) and 260-fold higher than that of l-Hcys (k _cat/K_m^l-Hcys = 0.027 ± 0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.

Full text of DOI:10.1016/j.bbapap.2013.11.012

A Role for Glutamate-333 of Saccharomyces cerevisiae Cystathionine γ-
Lyase as a Determinant of Specificity
Emily M.S. Hopwood, Duale Ahmed, Susan M. Aitken
PII:
DOI:
Reference:
S1570-9639(13)00418-4
doi: 10.1016/j.bbapap.2013.11.012
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Received date:
Revised date:
Accepted date:
11 July 2013
17 November 2013
21 November 2013
Please cite this article as: Emily M.S. Hopwood, Duale Ahmed, Susan M. Aitken, A Role
for Glutamate-333 of Saccharomyces cerevisiae Cystathionine γ-Lyase as a Determinant
of Specificity, BBA - Proteins and Proteomics (2013), doi: 10.1016/j.bbapap.2013.11.012
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ACCEPTED MANUSCRIPT
A Role for Glutamate-333 of Saccharomyces cerevisiae Cystathionine γ-Lyase as a
Determinant of Specificity.
*
Emily M.S. Hopwood, Duale Ahmed and Susan M. Aitken
Department of Biology, Carleton University, Ottawa, Canada, K1S 5B6.
*
5
To whom correspondence should be addressed. Telephone: (613) 520-2600. Fax: (613)
20-3539. E-mail: susan_aitken@carleton.ca
RUNNING TITLE: E48 and E333 OF YEAST CYSTATHIONINE γ-LYASE.
TOTAL MANUSCRIPT PAGES: 35
ABBREVIATIONS: AAT, aspartate aminotransferase; ACCS, 1-aminocyclopropane-1-
carboxylate synthase; AMPSO, N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-
hydroxypropanesulfonic acid; bicine, 2-(Bis(2-hydroxyethyl)amino)acetic acid; CAPS, 3-
(
Cyclohexylamino)-1-propanesulfonic acid; CBL, cystathionine -lyase; CGL,
cystathionine -lyase; CGS, cystathionine -synthase; L-Cth, L-cystathionine; DTNB,
,5’-Dithiobis(2-nitrobenzoic acid); L-Hcys, L-homocysteine; HO-HxoDH, D-2-
5
hydroxyisocaproate dehydrogenase; IPTG, Isopropyl--D-thiogalactopyranoside; LDH,
L-lactate dehydrogenase; MGL, methionine γ-lyase; NADH (reduced form), β-
nicotinamide adenine dinucleotide; Ni-NTA, Ni-nitrilo triacetic acid; L-OAS, O-acetyl-L-
serine; L-OSHS, O-succinyl-L-homoserine; OASS, OAS sulfhydrylase; PLP, pyridoxal
5
′-phosphate; TAPS, N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid.
1

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1
. ABSTRACT
Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of L-cystathionine (L-Cth),
producing L-cysteine (L-Cys), α-ketobutyrate and ammonia, in the second step of the
reverse transsulfuration pathway, which converts L-homocysteine (L-Hcys) to L-Cys.
Site-directed variants substituting residues E48 and E333 with alanine, aspartate and
glutamine were characterized to probe the roles of these acidic residues, conserved in
fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces
cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine
substitutions of E333 is increased by 0.4-1.2 pH units, likely due to repositioning of the
cofactor and modification of the pK of the pyridinium nitrogen. The pH profile of
a
yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect
of substituting E48, E333 or both residues is the 1.3-3, 26-58 and 124-568-fold reduction,
L-Cth
respectively, in the catalytic efficiency of L-Cth hydrolysis. The Km
of E333
L-OAS
substitution variants is increased ~17-fold, while Km
is within 2.5-fold of the wild-
type enzyme, indicating that residue E333 interacts with the distal amine moiety of L-Cth,
which is not present in the alternative substrate O-acetyl-L-serine. The catalytic efficiency
L-OSHS
m
of yCGL for α,γ-elimination of O-succinyl-L-homoserine (k /K
= 7 ± 2), which
cat
possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of
L-Cth
m
the physiological L-Cth substrate (k /K
= 2100 ± 100) and 260-fold higher than that
cat
L-Hcys
of L-Hcys (k /Km
= 0.027 ± 0.005), which lacks both distal polar moieties. The
cat
results of this study suggest that the glutamate residue at position 333 is a determinant of
specificity.
2

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2
. INTRODUCTION
Natural and specialty amino acids comprise a multi-billion-dollar market with 5-
% growth annually and an expanding range of uses in the chemical, pharmaceutical,
7
cosmetic and agri-food industries [1,2]. Unnatural amino acids also expand the scope of
biocatalytic applications of enzymes [3]. Both areas rely on the engineering of amino acid
biosynthetic enzymes and pathways. This will be facilitated by elucidation of the
structure-function relationships that underlie substrate and reaction specificity among
enzymes catalyzing transformations of amino acids, which are typically dependent on the
catalytically versatile pyridoxal 5’-phosphate (PLP) cofactor. The enzymes of the
microbial transsulfuration pathways, which interconvert the sulfur-containing amino
acids L-cysteine (L-Cys) and L-homocysteine (L-Hcys), provide an ideal model system for
this purpose as they share common structural features, but catalyze distinct side-chain
rearrangements of similar amino-acid substrates (Figure 1) [4].
Cystathionine γ-lyase (CGL) is the second enzyme of the reverse transsulfuration
pathway, which converts the L-Hcys product of S-adenosylmethionine metabolism to L-
Cys (Figure 1). Comparison of the structures of Escherichia coli cystathionine -synthase
(
eCGS) and cystathionine β-lyase (eCBL), of the bacterial transsulfuration pathway,
Saccharomyces cerevisiae CGL (yCGL) and Trichomonas vaginalis methionine γ-lyase
tMGL) illustrates the structural similarity among the enzymes of the γ-subfamily of the
(
large and catalytically diverse fold-type I of PLP dependent enzymes. Although these
enzymes share only ~35% amino acid sequence identity, including several common
active-site residues, the r.m.s. deviation for the superposition of ~350 C atoms is 1.5 Å
α
[
5-8]. Typical of members of the γ-subfamily, these enzymes catalyze the α,γ-
3

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replacement (CGS) and α,β (CBL) or α,γ-elimination (CGL and MGL) of similar sulfur-
containing amino acid substrates (Figure 1).
The conformation and orientation of substrate(s) within the active site has been
proposed to be a determinant of reaction specificity among the enzymes of the γ-
subfamily [9,10]. However, these factors are subtle and not evident from comparison of
the available crystal structures. Multiple residues and structural features contribute to the
regulation of specificity and the roles of active-site residues, even those conserved
between enzymes with distinct function, are dependent on the context of the specific
enzyme [4,11,12]. The pseudosymmetric L-Cth substrate of eCBL and yCGL must be
bound in distinct orientations in the active site to enable hydrolysis via α,β versus α,γ-
elimination mechanisms. Roles for residues E48 and E333 of yCGL (Figure 2),
conserved as acidic residues in eCGS (D45 and E325) and replaced by aromatic residues
in eCBL (F55 and Y338), as determinants of reaction specificity were proposed based on
structural comparison [5,6]. Farsi and coworkers subsequently observed that
interconversion of these two residues in eCBL, eCGS and yCGL reduces wild-type
activity but is not sufficient to result in a corresponding modification of specificity [13].
Similarly, the exchange of a pair of ~25-residue structurally distinct regions situated at
the mouth of the active site and in proximity to residues E48 and E333, via construction
of chimeric variants of eCBL and eCGS, also drastically diminishes the native reaction
without an observable increase in the in vivo targeted activity [12]. A novel role for
residues D45 and E325 of eCGS as determinants of reaction specificity was recently
described by Jaworski et al., as substitution with alanine and asparagine or glutamine,
respectively, enables the deamination of alanine [11]. These studies demonstrate the
4

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complexity of the system and illustrate the challenge of identifying the determinants of
specificity. This study builds on our earlier work focusing on structure-function
relationships among the structurally similar, but mechanistically distinct members of the
γ-subfamily. We describe the characterization of a series of 9 site-directed variants of
yCGL, which provide insight into the roles of active-site residues E48 and E333 [12,13].
5

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3
. MATERIALS AND METHODS
.1. Reagents. L-Cth [S-(2-amino-2-carboxyethyl)-L-homocysteine], L-Cys, L-Hcys, O-
3
acetyl-L-serine (L-OAS), β-nicotinamide adenine dinucleotide (β-NADH, reduced form)
and L-lactate dehydrogenase (LDH) were purchased from Sigma-Aldrich (Oakville,
Ontario). L-Hcys was prepared from the thiolactone as in Kashiwamata and Greenberg
[
14]. 5,5’-Dithiobis(2-nitrobenzoic acid) (DTNB) was obtained from Pierce and nickel-
nitriloacetic acid (Ni-NTA) resin was from Qiagen (Toronto, Ontario). 2-(Bis(2-
hydroxyethyl)amino)acetic acid (bicine), N-[Tris(hydroxymethyl)methyl]-3-
aminopropanesulfonic acid (TAPS), N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-
hydroxypropanesulfonic acid (AMPSO) and 3-(Cyclohexylamino)-1-propanesulfonic
acid (CAPS) were purchased from Fisher Scientific (Ottawa, Ontario).Oligonucleotide
primers were synthesized by Integrated DNA Technologies (Coralville, Iowa) and site-
directed variants were sequenced by BioBasic (Markham, Ontario) prior to expression.
The D-2-Hydroxyisocaproate dehydrogenase (HO-HxoDH) coupling enzyme was
expressed and purified as described previously [15].
3
.2. Construction, expression and purification of site-directed mutants. Site-directed
mutants of yCGL, constructed via the overlap-extension polymerase chain reaction
method, were inserted into the pTrc-99aAF plasmid. The amino-terminal, 6-His tag and
linker encoded by this vector enables affinity purification of the expressed enzymes and
does not alter the kinetic parameters of yCGL [13]. Wild-type and site-directed variants
of yCGL were expressed in E. coli strain ER1821metC::cat, in which the gene encoding
eCBL is replaced by chloramphenicol acetyltransferase, to prevent contamination with
6

ACCEPTED MANUSCRIPT
the β-lyase activity of the host eCBL enzyme, and purified, via Ni-NTA affinity
chromatography [13].
3
.3. Determination of steady-state kinetic parameters. Enzyme activity was measured in
a total reaction volume of 100 μL at 25°C and detected on a Spectramax 340 microtiter
plate spectrophotometer (Molecular Devices). The assay buffer was composed of 50 mM
potassium phosphate, pH 7.2, containing 20 μM PLP. Hydrolysis of L-Cth was monitored
-1
-1
via reaction of DTNB (ε412 = 13 600 M cm ) with the free thiol group of the product
[
13]. The α,β-elimination of L-Cys and L-OAS and the α,γ-elimination of L-Hcys were
detected via continuous, coupled assays in which the pyruvate and α-ketobutyrate
products are reduced by LDH (3.5 μM) and HO-HxoDH (3.4 μM), respectively, with
-1
-1
concomitant oxidation of NADH (ε340 = 6,200 M cm ) [16,17]. The concentration of
yCGL enzyme in the assay was between 0.45 – 19.5 μM, depending on the activity of the
specific site-directed variant. A background reading was recorded in all cases before the
reaction was initiated by the addition of yCGL enzyme. Data were fitted to the Michaelis-
Menten equation to obtain values of kcat and K for the hydrolysis of L-Cth and L-OAS
m
and k /K was obtained independently from equation 1. For site-directed variants for
cat
m
which saturation was not observed, including the hydrolysis of L-Hcys, k /K values
cat
m
were obtained by linear regression, based on the assumption that K >> [substrate]. The
m
L-Cys hydrolysis data were fitted to equation 2, which modifies the Michaelis-Menten
L-Cys
L-Cys
equation to incorporate the Ki
term for substrate inhibition by L-Cys, and k /K
cat m
values were obtained independently from equation 3.
7

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kcat

S

v
Km

(1)
S

E

1

Km
v
k 
 
S
cat


E




LCys
S
Ki
(2)
Km


S

1
LCys


kcat

S
 
v
Km

(3)

E





S


S
Ki
1

1


LCys
Km
3
.4. Evaluation of the pH dependence of yCGL. The pH dependence of L-Cth hydrolysis
by yCGL was determined using the continuous DTNB assay in a three-component buffer,
comprised of 50 mM MOPS (pK = 7.2), 50 mM bicine (pK = 8.3) and 50 mM proline
a
a
(
pK = 10.7) [18,19]. The kinetic parameters were determined at a range of pH values
a
between pH 6.4-10 in the presence of 20 M PLP, 2 mM DTNB, 0.45 – 19.5 μM yCGL
(depending on the activity of the site-directed variant) and 0.01-5.5 mM L-Cth. The
L-Cth
m
k /K
cat
versus pH data were fitted to the bell-shaped curve described by equation 4,
max
L-Cth
where k /Km is the upper limit for k /K
m
at the pH optimum [16].
cat
cat
kcat
max
Km
kcat
(4)

pKa1 pH
pH  pKa 2
Km
110
10
8

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3
.5. Spectrophotometric titration of the internal aldimine. The internal aldimine was
titrated versus pH with 20 μM enzyme in 5 mM TAPS (pK = 8.4; pH 6.70) containing
a
0
.5 M KCl as a starting point. The pH was increased by successive additions of 0.5 M
AMPSO (pH 10.6; pK = 9.0) at pH <9.0, 0.5 M CAPS (pH 11.5; pK = 10.4) between
a
a
pH 9.0 and 10.5, and 1.0 M NaOH at pH >10.5. The pH of the solution was determined
prior to each absorbance measurement [16]. Spectra were recorded on an HP 8453
spectrophotometer. The data recorded at 390 and 421 nm were fitted to equations 5 and 6,
respectively:
A  A
1
2
A 
 A2
pHpK
spec
1
1
10
(5)
A  A
1
2
A 
 A2
pK pH
(6)
spec
10
where A1 and A2 are the high and low absorbance limits at a particular wavelength,
respectively.
9

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4
. RESULTS
The nine single and double site-directed substitution variants targeting residues
E48 and E333 of yCGL were purified to homogeneity via Ni-NTA affinity
chromatography. The proteins are soluble, with yields between 2.7-6 milligrams of
protein obtained per litre of culture. Hydrolysis of the physiological L-Cth substrate as
well as the α,β-elimination of L-Cys and O-acetylserine (OAS) is detectable for all
variants (Tables 1 and 2). The α,γ-elimination of L-Hcys was also assayed. The enzymes,
including wild-type yCGL, are not saturated within the solubility limit of this substrate.
This, in combination with the low catalytic efficiency of the wild-type enzyme for
L-Hcys
hydrolysis of L-Hcys (k /Km
= 0.027 ± 0.005), which is 78,000-fold lower than for
cat
L-Cth
the hydrolysis of L-Cth (k /Km
= 2100 ± 100; Table 1), precludes meaningful
cat
comparison of kinetic parameters for L-Hcys.
4
.1. Comparison of yCGL with eCBL. Although yCGL and eCBL both catalyze the
L-Cth
hydrolysis of L-Cth, the k /Km
of eCBL is 90-fold greater than that of yCGL (Table
cat
1
). The pH profiles of both enzymes are bell shaped, with distinct pH optima of 7.2-8.0
and 8.5-9.5 for yCGL and eCBL, respectively (Figure 3). The value of pK (yCGL = 8.1
a1
±
0.4; eCBL = 8.28 ± 0.06) is within experimental error for both enzymes. In contrast,
pK of yCGL (7.1 ± 0.4) is 3 pH units lower than that of eCBL (10.20 ± 0.06). Spectral
a2
titration of eCBL and yCGL was performed to investigate a potential role for the
protonation state of the aldimine as a determinant of specificity, as exemplified by 1-
aminocyclopropane-1-carboxylate synthase (ACCS) and aspartate aminotransferase
(
AAT), which possess similarly conserved active sites but differ in the pK of the internal
a
10

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aldimines by 2.3 pH units [20]. The increase in absorbance at 290 nm above pH 9.5
(Figure 4), and corresponding decrease at 277 nm, observed for yCGL and eCBL likely
reflects tyrosine ionization (pK = 10.13). The 421 nm absorbance of yCGL and eCBL
a
shifts to 390 nm at pH > 9 and 10, respectively, within one pH unit of the observed 277-
to-290 nm transition. In contrast with the increase in pH optimum observed for yCGL-
E48A/E333A (Figure 3), the spectral titration of this variant (Figure 4) is identical to the
wild-type enzyme. The pK values for the loss of 421 nm absorbance are 10.04 ± 0.05
a
and 8.28 ± 0.03 and for the increase in absorbance at 390 nm are 11.08 ± 0.08 and 8.62 ±
0
.05 for eCBL and yCGL respectively. Although the ~2 pH unit difference between the
corresponding pK values of the two enzymes is consistent, the shift to 390 nm and the
a
mismatch between the 390 and 421 nm pK values for each suggests dissociation of the
a
cofactor rather than deprotonation of the aldimine in the context of the active site [16].
This is further supported by the lack of an isobestic point for eCBL, indicating that the
observed spectral shift does not reflect a two-state system (Figure 4).
L-Cth
4
.2. Effect of substitutions on the pH profile of yCGL. The pH optima of the k /K
cat m
versus pH profiles of the E48 substitution variants are within 0.4 pH units of the wild-
type enzyme (Table 1). Substitution of residue E333 with alanine (yCGL-E333A) or
glutamine (E333Q) results in an increase of 0.4 and 0.6 pH units. In contrast, replacement
with aspartate (E333D), which maintains the carboxylate moiety of the side chain but is
expected to shift its position as a result of the ~1 Å shorter side chain of aspartate, does
not change the pH optimum (Table 1). Substitution of both E48 and E333 with alanine or
L-Cth
glutamine results in increases of 0.7 and 1.2 units in the pH optimum of k /Km
, to
cat
11

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8
.4-9.2 and 7.9-8.7, respectively. The pH optimum of yCGL-E48D/E333D is also
increased, although by only 0.4 pH units (Table 1; Figure 3). The reduction of up to two
orders of magnitude in the observed versus maximal catalytic efficiency, resulting from
the reversed pK values (pK > pK ) of the wild-type and site-directed variants of yCGL,
a
a1
a2
precludes accurate estimations of the pK values for variants of these enzymes.
a
4
.3. Hydrolysis of L-Cth and alternate substrates. The effect on yCGL activity of
substituting residue E48 with alanine, aspartate and glutamine is minor as the kinetic
parameters for the hydrolysis of L-Cth, L-OAS and L-Cys are within 4-fold of the wild-
type enzyme (Tables 1 and 2). In contrast, the corresponding E333 substitutions result in
L-Cth
a ~17-fold increase in Km
and 2-3-fold decrease in kcat (Table 1). The catalytic
efficiency of the E333 replacement variants for the α,β-elimination of L-Cys is also
L-Cys
reduced, 2-13-fold, while Ki
is within 2.5-fold of the wild-type (Table 2). Substitution
of E333 with alanine, aspartate or glutamine alters the catalytic efficiency of L-OAS β-
elimination by less than 2.5-fold, compared to the wild-type enzyme (Table 2).
The double replacement variants of residues E48/E333 are not saturated within
L-Cth
m
the solubility limit of the L-Cth substrate, and k /K
is reduced by 570, 240, and 125-
cat
fold, respectively, compared to yCGL (Table 1). In contrast, the kinetic parameters for
hydrolysis of L-OAS by the E48/E333 double replacement variants are within 3-fold of
L-Cys
the wild-type enzyme (Table 2). The value of Km
is increased ~5-fold for all double
replacement variants of E48/E333 and the kcat of the E48D/E333D variant for L-Cys
hydrolysis is 5-fold lower than the wild type (Table 2).
12

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5
. DISCUSSION
Typical of the -subfamily of fold-type I, the PLP-dependent enzymes of the
transsulfuration pathways catalyze side chain rearrangements of similar amino acid
substrates, via α,β and α,γ-elimination and replacement reactions. Sharing a common
overall fold and several active-site residues, they present an effective model system for
investigation of the structure-function relationships underlying substrate and reaction
specificity in enzymes dependent upon the catalytically versatile PLP cofactor [4,12,13].
The current characterization of a series of 9 site-directed variants of two yCGL active-site
residues (E48 and E333) builds on our earlier work deciphering the subtle and complex
structure-function relationships of these structurally similar, but mechanistically distinct
enzymes [12,13].
5
.1. Role of E48 and E333 in substrate binding and specificity. Our analysis of a
representative selection of 16 fungal and animal CGL and 27 γ-proteobacterial CBL
amino acid sequences identified 65, 54 and 50 positions classified as at least 70%
conserved in CGL only (CGL-conserved, variable in CBL; e.g. yCGL-E343 and T349),
in CBL only (CBL-conserved, variable in CGL), and in both but as distinct residues
(conserved-different; e.g. E48 and E333 of yCGL correspond to F55 and Y338 of eCBL),
respectively. It is anticipated that a subset of these positions, acting in concert with a pair
of ~25-residue structurally distinct regions that frame the entrance of the active site, are
the primary determinants of reaction specificity. Investigation of the relative distances
between C4’ of the PLP cofactor and Cα of residues categorized as conserved or variable
positions shows that variable positions are common remote from the active-site,
13

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conserved-variable (CBL-conserved and CGL-conserved) and conserved-different
residues are primarily situated within 15-20 Å of the active site and positions identical in
both enzymes are more broadly distributed. Similarly, roles for residues outside of the
primary shell of the active-site, clustering between 10-27 Å and linked via contact
pathways of van der Waals and hydrogen-bonding interactions, were demonstrated by
Mendona and Marana using Spodoptera frugiperda β-glucosidase as a model system
[
21]. Among the 43 sequences aligned, the residue at position 333 (yCGL numbering) is
00% conserved as glutamate among CGL sequences and 100% conserved as an
1
aromatic residue (70% as tyrosine) in the CBL sequences. This provides indirect support
for the proposal of Huang and colleagues that increased hydrophobicity at this position
enables increased H S production from L-Cys, a substrate that allows only α,β-
2
elimination/replacement [22]. Roles in substrate binding and α,β versus α,γ-elimination
specificity have been proposed for E48 and E333 of yCGL, and the corresponding D45
and E325 of eCGS and F55 and Y338 of eCBL, as these residues are situated in the
access channel of the active-site. Interaction with the distal amino group of L-Cth and the
α-amino group of L-Cys was suggested for the acidic residues of yCGL and eCGS,
respectively, while hydrophobic packing with the L-Cth substrate was postulated for the
corresponding aromatic residues of eCBL [5-7]. Position 333 may also participate in the
definition of active-site architecture as residue yCGL-E333 interacts with T349, which is
situated in the ~25-residue structurally distinct, carboxy-terminal region. In contrast, the
corresponding eCBL-Y338 forms a hydrogen bond with K42, located in the nine-residue
insertion, unique to CBL, in the ~25-residue structurally distinct, amino-terminal region
[
7].
14

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The near-wild type kinetic parameters, for the hydrolysis of L-Cth, L-Cys, and L-
OAS, of the alanine, aspartate and glutamine substitution variants of E48 do not support a
direct role for this residue in reaction specificity or substrate binding (Tables 1 and 2).
Replacement of the corresponding D45 of eCGS with alanine or asparagine also results in
only 2-9-fold changes in kinetic parameters, but enables a minor transamination activity.
This observation suggests that, while this residue does not participate directly in substrate
binding, it may contribute to active-site architecture or substrate positioning to subtly
influence reaction specificity [11]. Substitution of the corresponding F55 of eCBL with
L-Cth
the aspartate, mimicking eCGS-D45, results in a 78-fold increase in Km
,
demonstrating the importance of active-site context [13].
Replacement of yCGL-E333 with alanine, aspartate or glutamine results in a ~17-
L-Cth
fold increase in Km
, confirming a role for this residue in L-Cth binding (Table 1). In
contrast, the kinetic parameters for L-OAS hydrolysis by the E333 substitution variants
are within 3-fold of the wild type enzyme (Table 2). This implies that yCGL-E333
interacts with the distal amino moiety of L-Cth. The L-OSHS substrate of eCGS, like L-
OAS, lacks the distal amino group of L-Cth and substitution variants of the corresponding
L-OSHS
eCGS-E325 also display similar 2–4-fold changes in Km
[11]. Although the
corresponding Y338 of eCBL interacts with K42, the kinetic parameters of the Y338F
and K42A replacement variants are similar to the wild-type eCBL enzyme, suggesting
that the Y338-K42 interaction and the hydrogen-bonding moieties of these side chains are
not required for efficient L-Cth hydrolysis [12,17]. As such, E333 plays unique role in
substrate binding, via interaction with the distal amino group of L-Cth, in the context of
yCGL. Therefore, we propose a role for this residue as a determinant of reaction
15

ACCEPTED MANUSCRIPT
specificity via positioning of the L-Cth substrate, in a manner distinct from eCBL. The
role of interactions with the distal portion of the substrate as a determinant of specificity
is supported by the observation that the catalytic efficiency of L-Hcys α,γ-elimination
L-Hcys
m
(
k /K
= 0.027 ± 0.005) is five orders of magnitude lower than the hydrolysis of L-
cat
L-Cth
Cth (k /Km
= 2100 ± 100) (Table 1). Similarly, the catalytic efficiency of yCGL for
cat
L-OSHS
m
α,γ-elimination of L-OSHS (k /K
= 7 ± 2), which possesses a distal carboxylate,
cat
but lacks an amino group, is 300-fold lower than that of the physiological L-Cth substrate
and 260-fold higher than that of L-Hcys, which lacks both distal polar moieties [13].
These observations provide support for the theory that positioning of substrate(s) within
the active site is an important determinant of reaction specificity among the enzymes of
the γ-subfamily [9,10].
Concomitant substitution of both E48 and E333 with alanine, aspartate, or
glutamine has a synergistic effect, in terms of the magnitude of perturbation of the kinetic
parameters of yCGL. The E48/E333 double replacement variants are not saturated within
L-Cth
m
the solubility limit of the L-Cth substrate and k /K
is decreased by up to 570-fold, at
cat
least an order of magnitude greater than the 1.3-3-fold and 26-58-fold reductions in
catalytic efficiency observed for the single substitution variants of E48 and E333,
respectively (Table 1). In contrast, the catalytic efficiency of the nine site-directed
variants for L-OAS and L-Cys hydrolysis are within 4-fold and 13-fold, respectively, of
the wild-type enzyme and the effect of double replacement, compared to E333 alone, is
minor (Table 2). Therefore, the role of the glutamate residue at position 333 as a
determinant of reaction specificity, in the context of yCGL, is enhanced by E48, as its
16

ACCEPTED MANUSCRIPT
concomitant substitution selectively impairs L-Cth hydrolysis, compared with the α,β-
elimination-specific substrates L-OAS and L-Cys.
5
.2. Effect of substitutions on yCGL pH dependence and the spectral properties of the
enzyme. Although eCBL, eCGS and yCGL share a common structure and several active-
site residues, the pH optima for the catalytic efficiency of the eCBL-catalyzed (8.5-9.5)
α,β-elimination of L-Cth is distinct from the 7.2-7.8 range for the α,γ-eliminations of L-
OSHS and L-Cth catalyzed by eCGS and yCGL, respectively (Table 1, Figure 3) [16,17].
The absorption spectra of eCBL and yCGL exhibit a λmax of ~421 nm, diagnostic for the
protonated form of the internal aldimine of the cofactor. Loss of this proton is
accompanied by a shift in the maximum of the spectrum of the PLP aldimine to 360 nm
[
23]. Modulation of the pK of the internal aldimine as a mechanism of regulating
a
specificity is exemplified by ACCS and AAT. The pK s of the aldimine and substrate,
a
which correspond to the two titrating groups of the bell-shaped catalytic efficiency versus
pH profile, are balanced such that that the values of 7.0 and 9.9, respectively, of AAT
reversed as 9.3 and 7.8, respectively, in ACCS to enable optimal activity at physiological
pH [20]. However, the shift in aldimine absorbance to 390 nm, similar to that of the free
cofactor, upon titration of eCBL and yCGL is likely due to dissociation of PLP [16].
Therefore, in contrast with AAT and ACCS, the aldimine of yCGL and eCBL, like that of
L-Cth
m
eCGS, cannot be assigned to either limb of the k /K
versus pH profile [16,20].
cat
Although the pH optima of yCGL and eCBL are distinct, differing by ~1.5 pH
units, the value of pK (yCGL = 8.1 ± 0.4; eCBL = 8.28 ± 0.06) is within experimental
a1
error for both enzymes. The titrating group of the acidic limb of the catalytic efficiency
17

ACCEPTED MANUSCRIPT
versus pH profile may correspond to yCGL-K203/eCBL-K210 as the ε-amino group must
be in the uncharged state to abstract the Cα proton of the substrate, initiating the ensuing
side-chain rearrangements. The pK of this lysine residue, the common catalytic base of
a
PLP-dependent enzymes catalyzing transformations of amino acids, may be reduced from
1
0.5 to less than 5 in the context of the active site, as exemplified by pig heart AAT
[
24,25]. The pK value of ~8.2 observed for yCGL and eCBL is similar to that reported
a1
for the corresponding K41 of Salmonella typhimurium O-acetylserine sulfhydrylase
[
26,27].
In contrast with the common pK value, the basic limb of the pH profile of yCGL
a1
(
pK = 7.1 ± 0.4) is 3 pH units lower than that of eCBL (pK = 10.20 ± 0.06). The 90-
a2
a2
fold lower catalytic efficiency of yCGL, compared to eCBL, may be due to the ~1 pH
unit lower value of pK , compared to pK , as the observed catalytic efficiency of yCGL
a2
a1
max
4
is an order of magnitude lower than the theoretical maximum (k /Km = 2 ± 1 x 10 ) at
cat
L-Cth
m
5
the pH optimum. In contrast, the observed (k /K
= 1.3 x 10 ) value is within 1.3-
cat
max
m
5
fold of the theoretical maxmum for eCBL (k /K
= 1.63 ± 0.07 x 10 ). The catalytic
cat
L-OSHS
efficiencies of the α,γ-elimination of L-OSHS and L-Cth by eCGS (kcatE/Km
= 1350
-1 -1
L-Cth
-1 -1
±
90 M s ) and yCGL (kcatE/Km
= 2100 ± 10 M s ), respectively, are of similar
magnitude but an order of magnitude lower than the physiological α,γ-replacement of
L-OSHS
-1 -1
eCGS (kcatR/Km
= 49000 ± 9000 M s ), suggesting that a step following formation
of the β,γ-unsaturated ketimine intermediate, common to both mechanisms, is rate
limiting for α,γ-elimination [16,28]. The reduced catalytic efficiency of yCGL, compared
to eCBL, resulting from the reversed pK values (pKa1 > pK ) of the former, may
a
a2
represent an evolutionary trade off between catalytic efficiency and reaction specificity as
18

ACCEPTED MANUSCRIPT
eCBL is specific for the α,β-elimination mechanism, while yCGL catalyzes the α,β- and
α,γ-elimination of L-Cth [13].
The group responsable for pKa2 may be one of the two amino groups of the
substrate, as proposed for the hydrolysis of L-Cth by cystathionine β-synthase. Aitken and
Kirsch reported pK values of 8.54 ± 0.01 and 9.63 ± 0.01 for the amino groups of L-Cth
a
by direct titration with NaOH [29]. It is possible that the pK of one or both of these
a
groups is influenced by the active-site environment of eCBL and yCGL, particularly as
residue E333 of yCGL uniquely interacts with the distal amino group of L-Cth and is
replaced with the aromatic Y338 in eCBL. Alternatively, the titrating group may be the
pyridinium nitrogen of the cofactor, which is influenced by proximity to the adjacent
asparate (yCGL-D178, eCBL-D185) as well as interaction between O3’ of the cofactor
and the protein (Figure 2) [24]. A network of polar interactions connects N1 and O3’ of
the cofactor in eCBL but not yCGL as eCBL-W340, which forms a hydrogen bond with
PLP-O3’, is conserved as leucine (yCGL-L335) in fungal and animal CGL sequences.
The pK of the pyridinium nitrogen may be influenced by yCGL-E333 and the
a
corresponding eCBL-Y338 via modulation of the distance between N1 and the
carboxylate group of the adjacent aspartate as the 1.1-Å greater distance in yCGL
diminishes the ability of this residue to promote protonation of N1, thereby reducing its
pK relative to eCBL. Residues Y338 and W340 of eCBL and E333 and L335 of yCGL
a
also flank the serine residue (eCBL-S339/yCGL-S334) that plays an essential role in
positioning of the catalytic base (Figure 2). Although the effect of substitution with
aspartate on the pH optimum is minor, the catalytic efficiency of the E333D and
E48D/E333D enzymes is ~5 and ~10-fold reduced, at their pH optima, compared to the
19

ACCEPTED MANUSCRIPT
corresponding alanine and glutamine substitution variants. This suggests that the ~1 Å
difference in the length of the side chain of aspartate, compared to glutamate or
glutamine, results in binding of the substrate in a strained or less catalytically productive
conformation. The pH optima of the E48A and E48Q, E333A and E333Q variants are
increased 0.4-0.6 and 0.7 pH units, respectively. Replacement of both residues has an
additive effect, as demonstrated by the increase of 0.4, 0.7 and 1.2 units in the pH optima
aspartate, glutamine and alanine double substitution variants, respectively (Table 1;
Figure 3). The effect of E333 substitution is likely the result of loss of the positioning
interactions of this residue with T349 and the distal amino group of the substrate, thereby
resulting in a shift in the relative position of L335, in van der Waals contact with the PLP
ring, allowing the cofactor to shift and shortening of the (PLP)N1-D178 H-bond (Figure
2
). We propose that the conserved-different residues flanking yCGL-S334 (E333 and
L335) and the corresponding eCBL-S339 (Y338 and W340) contribute to the regulation
of reaction specificity via interactions that optimize the relative positions of the substrate,
cofactor and catalytic base to modulate the electronic properties of the PLP and allow or
restrict access of the catalytic base to the C4’ and Cβ positions of the cofactor and
substrate, respectively.
The enzymes of the transsulfuration pathways are attractive targets for the
development of novel therapeutics, including antimicrobial compounds (CGS and CBL -
unique to plants and bacteria) and modulators of H S production (CGL). As members of
2
the γ-subfamily of fold-type I of PLP-dependent enzymes they also comprise a useful
model system for studies exploring the molecular mechanisms that underlie specificity
among enzymes reliant on this catalytically versatile cofactor, with the goal of
20

ACCEPTED MANUSCRIPT
engineering PLP-dependent enzymes for biotechnological applications. Both of these
objectives require an in-depth understanding of the structure-function relationships that
are the basis for inhibitor or reaction specificity. This study has identified a unique role
for E333 in binding to the distal amino moiety of the physiological substrate as well
influencing the pH optimum of yCGL. These findings enhance our understanding of the
complex factors regulating reaction specificity in enzymes of the γ-subfamily and
confirm that modification of specificity will require multiple, simultaneous changes
[
4,12,13]. Given the catalytic versatility of the PLP cofactor and the central role of amino
acid metabolism to cellular homeostasis, it is not surprising that PLP-dependent enzymes
have evolved multiple residues and structural features for the regulation of specificity.
The observed immutability of restriction endonucleases, exemplified by BamHI and
BglII, which share structural features and a common core in their DNA recognition
sequences but are not easily interconverted, demonstrates that the challenge of identifying
the determinants of reaction specificity between structurally conserved enzymes is not
unique to PLP-dependent enzymes [30].
6
. ACKNOWLEDGEMENTS
This work was supported by the Natural Sciences and Engineering Research Council of
Canada.
21

ACCEPTED MANUSCRIPT
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Table 1. Kinetic parameters of L-Cth hydrolysis for wild-type eCBL and yCGL and site-
a
directed variants of yCGL.
-1
-1 -1
pH
optimum
Enzyme
k (s )
K (mM)
m
k /K (M s )
cat m
cat
L-Cth  L-Cys + α-ketobutyrate + NH (α,γ-elimination)
3
L-Cth  L-Hcys + pyruvate + NH (α,β-elimination)
3
b
5
eCBL
34.1 ± 0.6
1.51 ± 0.03
0.54 ± 0.01
1.4 ± 0.02
1.29 ± 0.04
0.78 ± 0.05
0.98 ± 0.04
0.48 ± 0.09
n.s
0.18 ± 0.01
0.71 ± 0.04
0.76 ± 0.04
0.87 ± 0.04
0.9 ± 0.1
12 ± 1
1.9 ± 0.1 x 10
(2.1 ± 0.1) x 10
(7.1 ± 0.3) x 10
8.5 – 9.5
7.2 – 7.8
7.6 – 8.4
3
2
yCGL
E48A
3
E48D
(1.59 ± 0.05) x 10 7.0 – 7.8
3
E48Q
(1.4 ± 0.1) x 10
65 ± 1
7.4 – 8.2
7.8 – 8.6
7.2 – 8.0
7.6 – 8.4
8.4 – 9.2
7.6 – 8.4
7.9 – 8.7
E333A
E333D
12 ± 1
82 ± 1
E333Q
13 ± 3
36 ± 2
E48A/E333A
E48D/E333D
E48Q/E333Q
n.s.
3.7 ± 0.1
8.8 ± 0.1
16.9 ± 0.1
n.s.
n.s.
n.s.
n.s.
a
Reaction conditions: 50 mM potassium phosphate, pH 7.2, 20 μM PLP, 2 mM DTNB,
.01-5.5 mM L-Cth and 0.45-19.5 μM yCGL, depending on the activity of the specific
0
L-Cth
enzyme variant. The data were fitted to the Michaelis-Menten equation and k /K
cat
m
was determined directly via equation 1 and error values correspond to the fit of the data
to the model [13]. n.s. indicates the enzyme is not saturated within the solubility limited
of the L-Cth substrate. The yCGL enzymes were assayed at pH 7.2, a pH at which the
activity of eCBL, which is specific for the α,β-elimination of L-Cth, is negligible (<5% of
the maximal activity at pH 8.5). In contrast, yCGL catalyzes both the α,β- and α,γ-
elimination of L-Cth [13]. Therefore, the lowest pH of the optimal range of yCGL,
furthest from the pH optimum of eCBL, was selected to assay the L-Cth hydrolysis
activity of this enzyme as the distinct pH optima of the 2 enzymes may be a determinant
L-Cth
m
of α,β- versus α,γ- reaction specificity. The k /K
of yCGL at pH 7.2 and 7.6 (in
cat
MOPS-bincine-proline buffer), the lowest point and midpoint of the optimum range of
L-Cth
-1 -1
the pH versus k /K
profile are 2180 ± 90 and 2700 ± 100 M s , respectively.
cat
m
b
values from ref [10].
28

ACCEPTED MANUSCRIPT
Table 2. Kinetic parameters of L-OAS and L-Cys hydrolysis for wild-type and site-
a
directed variants of yCGL.
-
1
L-Cys
b
-1 -1
Enzyme
k (s )
K (mM)
m
Ki
(mM)
k /K (M s )
cat m
cat
L-Cys  pyruvate + H S + NH3
2
3
yCGL
E48A
0.15 ± 0.01
0.09 ± 0.01
0.32 ± 0.04
0.18 ± 0.01
1.3 ± 0.5
0.09 ± 0.01
0.11 ±0.02
0.4 ± 0.1
0.18 ± 0.02
1.2 ± 0.6
1.8 ± 0.4
0.7 ± 0.2
0.5 ± 0.1
0.5 ± 0.1
0.4 ± 0.1
2.0 ± 0.3
3.1 ± 0.6
2.2 ± 0.6
5.7 ± 0.7
0.8 ± 0.4
1.1 ± 0.3
3 ± 1
(1.7 ± 0.2) x 10
2
(8 ± 1) x 10
2
E48D
(7.2 ± 0.8) x 10
3
E48Q
(1.02 ± 0.06) x 10
2
E333A
(8.1 ± 0.7) x 10
2
E333D
0.24 ± 0.04
0.5 ± 0.1
(1.3 ± 0.1) x 10
2
E333Q
E48A/E333A
(4.9 ± 0.3) x 10
2
0.11 ± 0.02
0.4 ± 0.1
0.9 ± 0.2
0.7 ± 0.2
(2.1 ± 0.1) x 10
E48D/E333D 0.029 ± 0.004
60 ± 10
2
E48Q/E333Q
0.16 ± 0.04
(3.1 ± 0.2) x 10
L-OAS  acetate + pyruvate + NH3
yCGL
E48A
0.0097 ± 0.0002
0.0142 ± 0.0004
0.0053 ± 0.0001
0.0124 ± 0.0002
0.0058 ± 0.0002
0.0045 ± 0.0002
0.0102 ± 0.0002
2.5 ± 0.3
1.5 ± 0.2
1.4 ± 0.1
4.4 ± 0.3
2.2 ± 0.3
0.9 ± 0.2
5.3 ± 0.4
2.9 ± 0.6
0.7 ± 0.2
1.5 ± 0.3
-
-
-
-
-
-
-
-
-
-
3.8 ± 0.4
10 ± 1
E48D
3.8 ± 0.3
2.8 ± 0.2
2.6 ± 0.3
5 ± 1
E48Q
E333A
E333D
E333Q
1.9 ± 0.1
1.6 ± 0.3
5 ± 1
E48A/E333A 0.0047 ± 0.0002
E48D/E333D 0.0035 ± 0.0002
E48Q/E333Q 0.0092 ± 0.0004
6 ± 1
a
Reaction conditions: 50 mM potassium phosphate, pH 7.2, 20 μM PLP, 0.75-150 mM L-
-1
-1
OAS or 0.04-3 mM L-Cys, 3.5 μM LDH, 1.5 mM NADH (ε340 = 6,200 M cm ) and 2.2-
.8 μM yCGL, depending on the activity of the specific enzyme variant. The L-OAS and
L-Cys data were fitted to the Michaelis-Menten equation and equation 2 and k /K was
9
cat
m
determined directly via equations 1 and 3, respectively.
b
Substrate inhibition is not observed for the hydrolysis of L-OAS over the range of
substrate concentrations tested (0.75-150 mM).
29