On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.abb.2011.01.023

Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the NAD(P)⁺-coupled oxidation of succinic semialdehyde (SSA) to succinate, the last step of the γ-aminobutyrate shunt. Mycobacterium tuberculosis encodes two paralogous SSADHs (gabD1 and gabD2). Here, we describe the first mechanistic characterization of GabD1, using steady-state kinetics, pH-rate profiles, ¹H NMR, and kinetic isotope effects. Our results confirmed SSA and NADP⁺ as substrates and demonstrated that a divalent metal, such as Mg²⁺, linearizes the time course. pH-rate studies failed to identify any ionizable groups with pK_a between 5.5 and 10 involved in substrate binding or rate-limiting chemistry. Primary deuterium, solvent and multiple kinetic isotope effects revealed that nucleophilic addition to SSA is very fast, followed by a modestly rate-limiting hydride transfer and fast thioester hydrolysis. Proton inventory studies revealed that a single proton is associated with the solvent-sensitive rate-limiting step. Together, these results suggest that product dissociation and/or conformational changes linked to it are rate-limiting. Using structural information for the human homolog enzyme and ¹H NMR, we further established that nucleophilic attack takes place at the Si face of SSA, generating a thiohemiacetal with S stereochemistry. Deuteride transfer to the Pro-R position in NADP⁺ generates the thioester intermediate and [4A-²H, 4B-¹H] NADPH. A chemical mechanism based on these data and the structural information available is proposed.

Full text of DOI:10.1016/j.abb.2011.01.023

Archives of Biochemistry and Biophysics 509 (2011) 90–99
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1)
from Mycobacterium tuberculosis
Luiz Pedro S. de Carvalho a, , Yan Ling , Chun Shen , J. David Warren , Kyu Y. Rhee
⇑
a
b
b
a,c
a
Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Ave. New York, NY 10065, USA
Department of Biochemistry and Milstein Chemistry Core Facility, Weill Medical College of Cornell University, 1300 York Ave, New York, NY 10065, USA
Division of Infectious Diseases, Department of Medicine, Weill Medical College of Cornell University, 1300 York Ave, New York, NY 10065, USA
b
c
a r t i c l e i n f o
a b s t r a c t
+
Article history:
Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the NAD(P) -cou-
pled oxidation of succinic semialdehyde (SSA) to succinate, the last step of the -aminobutyrate shunt.
Received 7 December 2010
and in revised form 30 January 2011
Available online 12 February 2011
c
Mycobacterium tuberculosis encodes two paralogous SSADHs (gabD1 and gabD2). Here, we describe the
first mechanistic characterization of GabD1, using steady-state kinetics, pH-rate profiles, H NMR, and
kinetic isotope effects. Our results confirmed SSA and NADP as substrates and demonstrated that a diva-
lent metal, such as Mg , linearizes the time course. pH-rate studies failed to identify any ionizable
1
+
Keywords:
2+
Succinic semialdehyde dehydrogenase
Aldehyde dehydrogenase
Chemical mechanism
Stereochemistry
Hydride transfer
Isotope effects
a
groups with pK between 5.5 and 10 involved in substrate binding or rate-limiting chemistry. Primary
deuterium, solvent and multiple kinetic isotope effects revealed that nucleophilic addition to SSA is very
fast, followed by a modestly rate-limiting hydride transfer and fast thioester hydrolysis. Proton inventory
studies revealed that a single proton is associated with the solvent-sensitive rate-limiting step. Together,
these results suggest that product dissociation and/or conformational changes linked to it are rate-lim-
iting. Using structural information for the human homolog enzyme and 1H NMR, we further established
that nucleophilic attack takes place at the Si face of SSA, generating a thiohemiacetal with S stereochem-
+
2
istry. Deuteride transfer to the Pro-R position in NADP generates the thioester intermediate and [4A- H,
B- H] NADPH. A chemical mechanism based on these data and the structural information available is
1
4
proposed.
Ó 2011 Elsevier Inc. All rights reserved.
Introduction
neurotransmitter in the central nervous system. Pharmacologic
modulation of GABA levels in humans is a validated therapeutic
strategy used for the treatment of some forms of anxiety and epi-
lepsy [2]. The role of the GABA shunt in bacteria and plants is less
understood. In these organisms, GABA has been associated with glu-
tamate metabolism, anaplerosis, and/or in antioxidant defense. In
plants, activity of the GABA shunt rapidly increases in response to
stress. In Arabidopsis thaliana, a genetic knockout of the mitochon-
drial SSADH displayed drastic phenotypes such as altered growth,
necrotic lesions on leaves and hypersensitivity to light, attributed
to inability to cope with reactive oxygen damage [3].
Succinic semialdehyde dehydrogenases (SSADHs)1 are ubiqui-
tous enzymes that use NAD(P) and water to oxidize succinic semi-
aldehyde (SSA) to succinate (Scheme 1). These enzymes differ from
other succinic semialdehyde dehydrogenases that use CoA-SH as a
nucleophile, generating succinyl-CoA instead of succinate [1]. In
mammals, these enzymes are involved in the turnover of c-amino-
butyric acid (GABA), the last step of the GABA shunt (Scheme S1).
GABA is directly formed from glutamate and is the chief inhibitory
+
SSADH (EC 1.2.1.16) enzymes are relatively poorly studied in
comparison to other dehydrogenases, and with respect to kinetic
and chemical mechanism. Nonetheless, SSADHs from A. thaliana,
rat and human brains, Drosophila melanogaster and Escherichia coli,
have been purified and partially characterized [4–7]. The A. thali-
ana SSADH has a MW of 53 kDa and exists as a tetramer in solu-
⇑
Corresponding author. Address: Division of Mycobacterial Research, MRC
National Institute for Medical Research, London NW7 1AA, UK.
E-mail address: lsd2001@med.cornell.edu (L.P.S. de Carvalho).
Abbreviations used: SSADH, succinic semialdehyde dehydrogenase; SSA, succinic
1
semialdehyde; CoA-SH, coenzyme A; GABA,
c-aminobutyric acid; NMR, nuclear
magnetic resonance; IPTG, isopropyl b-D-1-thiogalactopyranoside; TEA, triethanola-
mine; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; GabD1, M.
tuberculosis SSADH1; MES, 2-(N-morpholino)ethanesulfonic acid; CHES, 2-(N-cyclo-
hexylamino)ethanesulfonic acid; TAPS, N-tris[hydroxymethyl]methyl-2-aminopro-
panesulfonic acid; PIPES, piperazine-N-N -bis[2-ethanesulfonic acid]; CAPS, 3-
cyclohexylamino)propanesulfonic acid; KIE, kinetic isotope effect; SIE, solvent
tion. Steady-state kinetic analysis indicated high specificity for
+
SSA and NAD as substrates, with KmSSA = 15
lM and KmNAD+
=
1
30 lM [6]. Interestingly, ATP was shown to allosterically inhibit
0
+
activity and affect NAD binding, suggesting regulation of enzyme’s
(
kinetic isotope effect; EIE, equilibrium isotope effect.
activity by the energy status of the cell [7]. Rat brain SSADH
0
003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2011.01.023

L.P.S. de Carvalho et al. / Archives of Biochemistry and Biophysics 509 (2011) 90–99
91
2
aldehyde dehydrogenase (ALDH). As shown in Fig. 1a, HsaSSADH
has only two residues optimally positioned for catalysis, Glu306
and Cys340. SSA is held in the active site mainly by its carboxylate,
which interacts by hydrogen bonding and/or electrostatics with
+
Arg213, Arg334 and Ser498. The ADP moiety of NAD is stabilized
by several residues including a group of hydrophobic residues,
Ile201, Ala 264, Gly268 and Leu292, which make a hydrophobic
pocket for the adenine, Lys228, Glu231 and Thr288 stabilize the ri-
bose, and the diphosphate hydrogen bonds with Ser285 and
Scheme 1.
+
displayed even lower KmSSA (2.5 lM), absolute specificity for NAD ,
and a compulsory kinetic ordered mechanism with NAD as the
+
+
Thr288. The remaining piece of the NAD could not be properly
first substrate to bind to the enzyme and NADH the last product
to dissociate [4]. H NMR studies indicate that SSA exists in solu-
modeled.
1
The human pathogen Mycobacterium tuberculosis has all pre-
dicted homologs of the GABA shunt enzymes, and two paralogous
SSADHs, encoded by gabD1 (Rv0234c) and gabD2 (Rv1731) genes
[10]. Both gene products were shown to catalyze oxidation of
SSA to succinate, but substrate specificity was not studied further
[11]. In M. tuberculosis, the most likely physiological route for pro-
duction of SSA is through the GABA shunt enzymes glutamate
decarboxylase and GABA transaminase. We recently showed that
the Rv1248c-encoded 2-hydroxy-3-oxoadipate synthase does not
tion as a 1:1 mixture of two forms, its aldehydic and gem-diol
forms. Saturation transfer difference NMR experiments demon-
strated that only the aldehydic form was competent to bind to
the E. coli and D. melanogaster SSADHs [5].
The three-dimensional structures of human and E. coli SSADHs
(
HsaSSADH and EcoSSADH, respectively) were also recently deter-
+
mined by X-ray crystallography [8,9]. The NAD -utilizing HsaS-
SADH was shown to exist in both an oxidized and a reduced
form, involving an intramolecular disulfide bond between the cat-
alytic cysteine residue (Cys340) and an adjacent cysteine residue
generate SSA, and instead couples the decarboxylation of
a-keto-
glutarate to condensation with glyoxylate [12]. Interestingly, pri-
mary sequence analysis indicates that despite significant
sequence identity and similarity, both GabD1 and GabD2 lack the
commonly observed second cysteine residue (non-catalytic thiol)
in the catalytic loop (Fig. 1b), responsible for disulfide formation
in the human enzyme. This residue also appears to be conserved
in several other SSADH homologs [8]. Lack of this second cysteine
residue is predicted to drastically alter the properties of mycobac-
terial GabDs, especially the ones concerning the oxido-reduction of
the catalytic thiol, and conformational changes linked to NAD(P)+
binding and chemistry. The proposed catalytic cysteine and gluta-
mate residues are conserved (Cys319 and Glu285, GabD1 number-
ing). While gabD2 appears to be nonessential, based on transposon
mutagenesis data, no knockout or transposon mutants of gabD1
have been described to date, implying that it might be essential
for growth [13]. Moreover, no kinetic or mechanistic studies have
been reported for either enzyme to establish their activity as
(
Cys342), within a ‘‘catalytic loop’’. The sensitivity of the catalytic
thiol to oxidation and disulfide formation was interpreted to repre-
sent a potential redox switch. That is, once oxidized, the catalytic
+
loop was proposed to close the active site, allowing NAD to bind
only in a conformation that does not allow catalysis. Upon disulfide
reduction, this same catalytic loop was proposed to reorient, allow-
ing NAD+ to assume a productive conformation. Although the ac-
tive sites of HsaSSADH and EcoSSADH are very similar, with
identical orientation of most of the catalytic residues and an addi-
tional cysteine residue in the catalytic loop, the EcoSSADH was
found to be sensitive to oxidation. However, this modification
was not reversed by addition of DTT, as observed with the human
homolog. In addition, size exclusion chromatography and crystal-
lographic data demonstrated that the oligomerization state of
HsaSSADH and EcoSSADH was tetrameric. As expected, the struc-
tures of the two SSADHs share a general fold of class 1 and class
Fig. 1. Active site of the human SSADH and primary sequence comparison with GabD1. (a) Active site of Cys340Ala mutant of human SSADH (yellow) in complex with SSA
PDB_ID 2W8Q), and the reduced form of the wild-type enzyme (pink) without SSA (PDB_ID 2W8O), at 2.4 Å and 3.4 Å resolution, respectively. Potential hydrogen bonds of
(
SSADH with SSA are shown as dashed lines, with corresponding distances in Å. Image was generated using PyMol. (b) Alignment of human SSADH and M. tuberculosis GabD1.
Amino acids highlighted in red are identical in both enzymes (31.5% identity) and amino acids highlighted in blue are strongly similar (20% strong similarity). Green boxes
+
indicated residues involved in NAD binding, blue boxes indicate residues involved in SSA binding, and red boxes indicate residues that are involved in catalysis (Cys319 and
Glu285, GabD1 numbering). Alignment was performed using ClustalW, with Blosum matrix, and the default parameters.

9
2
L.P.S. de Carvalho et al. / Archives of Biochemistry and Biophysics 509 (2011) 90–99
+
SSADHs, rather than broad specificity ALDHs. The identity and role
of mycobacterial GabDs in aldehyde detoxification, glutamate
metabolism and energy generation, through production of succi-
nate, thus remains undefined.
of SSA at a fixed saturating concentration of NADP (200 lM) and
2
+
Mg (10 mM). The following buffers were used at the indicated
pH ranges: MES (pH 5.73–6.2), PIPES (pH 6.0), HEPES (pH 6.5–8.3),
TAPS (pH 8.1–8.4), and CHES (pH 8.6–9.45), allowing overlap
[15,16]. The pH values reported are the final ones, determined with
the reaction mixtures after the initial rate was recorded, allowing
correction for changes due to buffer dilution and pH changes caused
Here, we report the first mechanistic characterization of the M.
tuberculosis succinic semialdehyde dehydrogenase 1 (GabD1) en-
zyme. We specifically characterize the substrate specificity of
GabD1 and its chemical mechanism, including the stereochemical
course of the reaction, using steady-state kinetics, pH-rate studies,
2
by the added substrates, MgCl and enzyme solutions.
1
H NMR spectroscopy, kinetic isotope effects.
Inactivation studies with H O
2
2
Experimental procedures
2 2
The oxidation of GabD1 by H O was studied by following the
loss of enzymatic activity upon exposure to H
2 2
O . 225 nM solution
Materials
of GabD1 containing 1 mM H O was incubated for up to 40 min, at
2
2
4
°C. Aliquots were taken every 10 min and enzymatic activity was
All chemicals were obtained from Sigma, unless otherwise sta-
ted. Nickel-NTA resin was from Novagen. Chromatographic col-
umns were purchased from GE. SSA solutions were prepared
from commercially available SSA, titrated, and stored at ꢀ80 °C.
assayed. Reaction mixtures contained 100 mM HEPES pH 7.4,
+
10 mM MgCl , 100
2
l
M SSA and 200
Stereochemistry of hydride transfer
To determine the stereospecificity of the hydride transfer cata-
lM NADP .
Trypsinolysis/LC–MS of GabD1
2
1
lyzed by GabD1 we used [ H]SSA and H NMR analysis of the reac-
tion product, as described elsewhere [17]. In short, the reaction
mixtures contained 50 mM KPi pH 7.4, 10 mM MgCl
NADP , 1 mM [ H]SSA, and 3 lM enzyme in D O. Reaction progress
was followed by H NMR, as described below. Each spectrum aver-
ages 128 scans, obtained during 11 min.
In order to identify whether or not GabD1 was able to react with
SSA we performed mass measurements with GabD1 in the pres-
ence or absence of ligands. Reaction mixtures contained (1)
2
, 1 mM
+
2
2
1
0
l
M GabD1 in buffer A (50 mM KPi, 10 mM MgCl
GabD1 in buffer A containing 100 M SSA and (3) 10
in buffer A containing 100 M SSA and 100 M NADPH. Samples
2
), (2) 10
l
M
1
l
lM GabD1
l
l
were trypsinized and peptides were separated and analyzed by
LC–MS using an LTQ-Orbitrap (Rockefeller University’s Proteomics
Resource Center).
Kinetic isotope effects
Kinetic isotope effects (KIEs) for the GabD1-catalyzed reaction
were determined in 100 mM HEPES pH 7.4, at saturating concentra-
Determination of bound ligands
2+
tions of Mg . Primary deuteriumKIEs on V and V/K were determined
1
2
using [ H]SSA and [ H]SSA, in the presence of saturating concentra-
Due to hysteresis observed with GabD1 (pronounced lag phase
under steady-state conditions), we sought to test our enzyme
preparation for heat-labile ligands, of which slow dissociation
+
1
tions of NADP , or at a fixed saturating concentration of [ H]SSA and
H]SSA and variable concentrations of NADP . Solvent isotope ef-
2
+
[
fects (SIEs) on V and V/K were determined in 85% D O or 0% D O,
2
2
could cause the lag observed in the time-courses. 200
l
L solution
1
+
using [ H]SSA in the presence of saturating concentrations of NADP ,
of 85 M GabD1 was boiled for 5 min to denature protein and re-
l
1
or at a fixed saturating concentration of [ H]SSA and variable con-
lease potential small molecules. This solution was cleared by cen-
trifugation followed by filtration on an YM10 filter (Millipore).
Filtered supernatant was probed by UV–vis spectroscopy and mass
spectrometry for small molecules. For LC–MS analysis, sample was
extracted with acetonitrile/methanol/water (2:2:1, v/v/v) and sep-
arated and analyzed using a Congent Silica Hydride Type C column
using an Agilent 6220 accurate mass time-of-flight mass spectrom-
eter, as described elsewhere [12].
+
centration of NADP . Multiple KIEs on V and V/K were determined
1
2
in D
2
O using [ H]SSA and [ H]SSA, in the presence of saturating con-
+
centrations of NADP , or at a fixed saturating concentration of
1
2
+
[
H]SSA and [ H]SSA and variable concentration of NADP .
Data analysis
Data were fitted using the nonlinear, least-squares, curve-fitting
programs of SigmaPlot 2000 for Windows, version 6.00. The quality
of the fit to the equations used was confirmed by plotting the
residuals of y versus x, and looking for nonrandom distribution,
which indicates systematic error [18]. Errors were propagated as
described in Skoog and West for indeterminate errors [19].
Saturation curve showing a non-zero Y intercept was fitted to
equation
Measurements of enzymatic activity
Enzymatic activity was measured by following the increase in
+
absorbance at 340 nm resulting from the reduction of NADP
ꢀ1
ꢀ1
(
e
340 = 6220 M cm ). Samples were pre-equilibrated at 37 °C
and the reaction was initiated by addition of enzyme, typically
5 nM. Initial velocity was estimated from the initial linear portion
of the time-courses. Unless stated otherwise, all experiments were
4
m
¼
m0 þ ½VA=ðA þ KÞꢁ
ð1Þ
2
+
performed in the presence of saturating concentrations of Mg
10 mM MgCl ). Viscosity experiments were carried out with 9%
where is the velocity in the absence of activator, V is the maximal
m
0
(
2
velocity, A is the concentration of activator, and K is the concentra-
tion of activator that gives half-maximal activation (Kact). Individual
saturation curves showing linear substrate inhibition were fitted to
glycerol (w/v) as viscogen, as described elsewhere [14].
pH-rate studies
2
m
¼ VA=½K þ A þ ðA =K
i
Þꢁ
ð2Þ
The pH dependence of kcat/KNADP+ and kcat were studied by vary-
+
ing the concentration of NADP at a fixed concentration of SSA
where V is the maximal velocity, K is the Michaelis constant for sub-
strate (K ), A is the concentration of substrate, and K is the appar-
ent inhibition constant for substrate A. Individual saturation curves
(
50
l
M) and a fixed saturating concentration of Mg2+ (10 mM). The
pH dependence of kcat/KSSA was studied by varying the concentration
m
i

L.P.S. de Carvalho et al. / Archives of Biochemistry and Biophysics 509 (2011) 90–99
93
displaying partial substrate inhibition were fitted to Eq. (3), which
is a general equation for nonhyperbolic kinetics
2
2
m
¼ ðaA þ bA Þ=ð1 þ cA þ dA Þ
ð3Þ
where a, b, c, and d are complex functions of rate constants and con-
centration of the nonvaried substrate and therefore have no physi-
cal meaning. Data plotted in the Lineweaver–Burke format was
fitted to
1
=m
¼ ðA þ KÞ=VA
ð4Þ
Individual saturation curves were fitted to Eq. (5), which is the re-
ciprocal of Eq. (4)
m
¼ VA=ðA þ KÞ
ð5Þ
Primary deuterium and solvent kinetic isotope effects were fit-
ted to Eqs. (6)–(8) for isotope effects on V only, V/K only, or both V
and V/K, respectively
m
m
m
¼ VA=½K þ Að1 þ F
i
E
V
Þꢁ
ð6Þ
ð7Þ
ð8Þ
¼ VA=½Kð1 þ F
¼ VA=½Kð1 þ F
i
E
E
V=KÞ þ Aꢁ
i
V=KÞ þ Að1 þ F
i
E
m
Þꢁ
where F
i
is the fraction of the isotopic label and E
V
and EV/K are the
isotope effects minus one on V and V/K, respectively. Linear Vmax
proton inventory was fitted to
V
n
¼ V
n
½1 ꢀ n þ nðk
D
=k
H
Þꢁ
ð9Þ
where n is the atom fraction of deuterium, V
n
is the velocity in the
0
solvent with the atom fraction of deuterium n, V is the velocity in
H
2
O, and k /k is the isotope effect.
D
H
Results
General properties of GabD1
GabD1 progress curve is characterized by a pronounced lag
which precedes steady-state velocity (Fig. 2a). Steady-state veloc-
ity does not decrease until nearly all substrates have been con-
Fig. 2. General properties of GabD1-catalyzed oxidation of SSA. (a) Time-course of
the NADP -dependent oxidation of SSA catalyzed by M. tuberculosis GabD1. (b)
+
verted to products, indicating low K
m
values for substrates and
high K values for products. Although long incubations led to a
i
Stoichiometry of the GabD1-catalyzed oxidation of SSA. Reaction mixtures
+
+
+
contained 100 mM HEPES pH 7.4, 10 mM MgCl , 100
lM NADP , 90 nM GabD1
2
modest utilization of NAD as substrate (Fig. 2a), NADP is the pre-
+
and SSA at 10, 40, 100, 400, and 1000 lM. Reactions were followed for up to 30 min
ferred substrate. Also, high concentrations of NAD led to visible
to assure that completion was reached. Reactions were performed in duplicates.
Dashed line represents the maximum NADPH concentration that could be reached
assuming a 1:1 stoichiometry. (c) Activation of GabD1 by divalent metals. In the
absence of added divalent metals or in the presence of 1 mM EDTA the time course
displays hysteresis (lag to achieve steady-state velocity). Addition of saturating
precipitation, impeding further characterization. Of the aldehydes
tested, only SSA served as a substrate (Table 1). Other aldehydes,
such as formaldehyde, acetaldehyde and glutaraldehyde, were very
poor substrates and, at high concentrations, caused visible precip-
itation of GabD1, precluding further characterization.
concentrations of Mg2 , Mn , Ca , Co and Ni led to linearization of the time
+
2+
2+
2+
2+
courses and a slight increase of the steady-state initial velocity.
Pre-incubation of GabD1 with either substrate did not alter the
time-course (data not shown). In addition, no change in the reac-
tion rate was observed in the presence of CoA-SH and no succi-
nyl-CoA was detected by spectrophotometry and liquid
chromatography coupled to time-of-flight mass spectrometry
try. These results also indicate that GabD1 catalyzed oxidation of
SSA to succinate is irreversible under all experimental conditions
tested.
(
LC–MS) (data not shown), ruling out succinyl-CoA as product of
GabD1 is activated by divalent metals such as Ca2+, Mg2+, Mn2+
,
Co2+, and Ni (Fig. 2c) but not Cd or Zn (data not shown). This
divalent cation activation increased steady-state rates of GabD1
only modestly (2- to 3-fold) but linearized the time-course
2+
2+
2+
GabD1. To probe if GabD1 contained a tightly bound, heat-unstable
inhibitor, which could cause the observed lag in reaction progress,
we performed UV–vis spectroscopy and LC–MS analysis on the
supernatant of heat-denatured GabD1 samples. No difference be-
tween buffer and GabD1 samples was observed, indicating that
the lag observed was not caused by slow dissociation of a tightly
bound inhibitor (data not shown).
2
+
(Fig. 2c). By varying the concentration of Mg at saturating sub-
+
2+
strates (200
value of 0.30 ± 0.07 mM. More detailed analysis, using Mg , re-
vealed a 4-fold decrease in the K value for SSA and a 6-fold de-
value for NADP (Table 1 and Fig. 3). Addition of
lM NADP and 50 lM SSA), we observed a Kact Mg
2+
m
+
To confirm the irreversibility of the reaction and proposed stoi-
chiometry, we quantified the amount of NADPH in a mixture con-
taining increasing concentrations of SSA but fixed quantity of
crease in the K
m
an excess of divalent metal chelator (5 mM EDTA) did not alter
the activity of GabD1 (Fig. 2c and data not shown), indicating an
absence of loosely associated divalent metal in our enzyme prepa-
ration and reaction mixture. Prolonged pre-incubation of GabD1
with EDTA (4 h) prior to activity measurement also did not alter
NADP+ (100
lM). As seen in Fig. 2b, the amount of NADPH pro-
duced is identical to the initial concentration of SSA until 100
l
M
+
(
concentration of NADP present). This confirms a 1:1 stoichiome-

9
4
L.P.S. de Carvalho et al. / Archives of Biochemistry and Biophysics 509 (2011) 90–99
Table 1
Steady-state kinetic parameters for GabD1.a
[Mg2+] (mM)
k
cat (s )b
ꢀ1
K
(
l
M)b
k
cat/K (M
ꢀ1 ꢀ1
s
)
K
(
l
M)b
Equation used
(2)
(3) and (4)
(5)
(5)
Variable ligand
Fixed substrate concentration (
lM)
m
i
5
SSA
200
200
50
–
10
–
1.9 ± 0.2
4.7 ± 0.1
1.3 ± 0.1
2.7 ± 0.1
13.3 ± 2.6
3.0 ± 0.2
61.3 ± 7.5
9.2 ± 1.0
1.4 ± 0.2 ꢂ 10
1.6 ± 0.1 ꢂ 10
2.1 ± 0.1 ꢂ 10
2.9 ± 0.1 ꢂ 10
62.8 ± 12
ND
–
–
6
5
5
c
d
SSA
NADP+
NADP+
50
10
a
b
c
Reactions performed at pH 7.5 and 37 °C.
Values are mean of two experiments ± standard error obtained upon fitting the data to the appropriate equation.
ND = not determined.
d
Numbers displayed were derived from the fit of the uninhibited segment of the curve to Eq. (3). Fit of the entire data to Eq. (4) gave the following results: a = 0.78, b = 0.03,
c = 0.03 and d = 0.02.
Fig. 3. Activation of GabD1 by Mg2+. Effect of MgCl
on the initial velocity of GabD1.
2
Symbols represent data from duplicates ± standard error, and solid lines represent
the fit to Eq. (1). These results are representative of two independent experiments.
the time course (data not shown), indicating a similar lack of
slowly dissociating metal bound to our preparation of GabD1.
Mass spectrometry analysis of GabD1 in the absence and in the
presence of SSA, and SSA:NADPH indicates that thiol addition to
+
the aldehyde does not occur in the absence of NADP (data not
shown). Trypsinolysis of solutions containing GabD1, GabD1:SSA,
and GabD1:SSA:NADPH displayed identical peptide profiles show-
ing that the catalytic cysteine residue was not modified in any of
the above-mentioned conditions. These results are consistent with
the requirement of ternary complex formation, prior to chemistry.
Attempts to oxidize the catalytic cysteine residue and inactivate
GabD1 using H
nificant loss of catalytic activity, after up to 40 min of incubation of
GabD1 with 1 mM H at pH 7.4 (data not shown). This result
2 2
O were unsuccessful. That is, we observed no sig-
2 2
O
thus indicates that either the thiol is protonated, in the free en-
zyme and therefore more protected from oxidation, or it is inacces-
sible to hydrogen peroxide.
Fig. 4. GabD1 steady-state kinetics. (a) Titration of GabD1 with NADP+ at constant
SSA (50
M), in the presence (closed circles) or absence (open circles) of Mg2+
10 mM). Solid lines represent the fit to Eq. (4). (b) Titration of GabD1 with SSA at
l
(
+
constant NADP (200
l
M), in the presence (closed circles) or absence (open circles)
of Mg2 (10 mM). Solid lines represent the fit to Eq. (2) (open circles) and Eq. (3)
solid circles). Open or closed symbols represent data from replicates ± standard
+
Steady-state kinetics
(
error. These results are representative of two independent experiments.
Initial velocities were determined by varying the concentration
of one substrate at a fixed, saturating concentration of the co-sub-
strate, and found to be proportional to the concentration of en-
In the presence of saturating Mg2+, substrate inhibition by SSA
was observed to be only partial, i.e. velocity decreased to a non-
zero value at saturating concentrations of SSA.
+
zyme. Kinetic parameters obtained for SSA and NADP , in the
presence or absence of saturating Mg2 are summarized in Table
+
+
1
. Saturation curves for GabD1 with NADP followed Michaelis–
Menten kinetics (Fig. 4 upper panel). In contrast, saturation curves
for SSA displayed nonhyperbolic response, showing an intermedi-
ate plateau (Fig. 4 lower panel). In the absence of Mg2 , SSA reac-
tion kinetics fitted well to a linear model of substrate inhibition,
pH-rate studies
+
We employed pH-rate studies to probe the role of general acid–
base chemistry on the reactions catalyzed by GabD1. Control
experiments were performed to confirm GabD1’s stability at the
2
+
but at saturating Mg , could not be fitted to the same equation.

L.P.S. de Carvalho et al. / Archives of Biochemistry and Biophysics 509 (2011) 90–99
95
[2
H]SSA, at pH 7.4 (Table 2). DV/K (1.8/1.5) were observed when
SSA was varied, indicating modest contribution of hydride transfer
to the catalytic rate of GabD1. The lack of a KIE on V/KNADP+ under
+
these conditions indicates that either NADP is a very sticky sub-
strate or that the kinetic mechanism is steady-state ordered with
+
NADP binding first. To differentiate between these two cases, we
D
measured the V/KNADP+ at different concentration of SSA. At lower
SSA concentrations, a normal KIE is observed, indicating that the ki-
+
netic mechanism is, in fact, random, and that NADP is a sticky sub-
strate. Further analysis of the dependence of the co-substrate on the
KIEs was not possible due to the complex kinetics and tight binding
D
of the substrates. Lack of V indicates that a step after the first irre-
versible step (hydrolysis), either product release or conformational
change linked to product release, is also partially rate-limiting.
Solvent kinetic isotope effects
SIEs were employed to study the reaction catalyzed by GabD1
D2O
at pH 7.4 (Table 2 and Figs. S1 and S2). Normal SIEs of
V (2.4–
D2O
+
2
.5) and
V/K (2.9–2.8) were observed when NADP was varied,
while no V/K was observed when SSA was varied. These results
suggest that a solvent-sensitive step is significantly rate-limiting
D2O
D2O
D2O
+
for the reaction catalyzed by GabD1. As both
V and
V/K
Fig. 5. pH dependence of GabD1 catalyzed NADP -dependent oxidation of SSA. The
cat and K values were determined at each pH by varying the concentration of one
substrate at a fixed saturating concentration of the other substrate, and saturating
k
m
are seen, it is likely that these effects reflect isomerization of free
enzyme to a form that allows NADP binding. Control experiments
+
concentrations of Mg2 . kcat/K
+
is the ratio of the values. Symbols are data and error
m
confirmed that these results are not explained by increased sol-
bars represent the standard error. Dotted lines represent the average of the data and
were added to aid visualization.
2
vent viscosity. Although it appears that SSA does not show a
D2O
V/K isotope effect, the low K
m
value for this substrate and its
limiting pH values used. GabD1 could not be assayed below pH 5.5,
due to precipitation and loss of activity (data not shown). As shown
in Fig. 5, no ionizable groups required for any rate-limiting chem-
istry, in the pH range of 5.5–10.0 could be detected. In addition, no
ionizable groups required for substrate binding were similarly de-
tected within this range. These results strongly suggest that the
complex kinetics make this result unreliable. To determine the
number of protons transferred during the solvent isotope-sensitive
D2O
step,
V proton inventory experiments were carried out. As seen
D2O
in Fig. S2, the linear correlation of the
V with the atom fraction
of D O indicates a single proton transfer during this solvent isotope-
2
sensitive step.
a
groups involved in substrate binding and chemistry have pK val-
ues out of the range analyzed and/or that additional pH-dependent
chemistry is not rate-limiting (see Discussion).
Multiple isotope effects
To determine the relative timing of the steps associated with
the primary deuterium KIEs and SIEs, multiple isotope effects were
Primary deuterium kinetic isotope effects
2
employed (Table 2). The use of [ H]SSA in the SIEs caused only a
D2O
To characterize the contribution of the hydride-transfer step to
the rate of the GabD1-catalyzed reaction, we conducted primary
deuterium kinetic isotope effects experiments, using [ H]SSA or
decrease in the
the
V/KNADP+ (Table 2 and Fig. S1). This decrease in
D2O
V/KNADP+ associated with deuteration of SSA indicates that
1
hydride transfer and proton transfer occur in separate steps (re-
D2O
viewed in [20]). The lack of change in the
V value (as well as
D2O value) indicates that an enzyme isomerization rather
than a chemical step is rate-limiting.
Table 2
D
in the
V
a
Primary deuterium and solvent kinetic isotope effects for GabD1-catalyzed reaction.
Parameter
Solvent
KIEb
Looking at the variation of the primary deuterium KIE as a func-
D
1
st exp.
2nd exp.
tion of deuterated solvent, we observed that the V/KSSA becomes
D
D
unity in D
O, while V/KNADP+ goes from unity in H
2
O to ca. 0.6 in
V
H
H
H
H
H
H
H
H
H
H
2
2
2
2
2
2
2
2
2
2
O
O
O
O
O/D
O/D
O/D
O/D
O/D
O/D
O
1
1
2
D
V/KSSA
1.8 ± 0.3
1
1.4 ± 0.2
2.4 ± 0.1
1
2.9 ± 0.9
2.2 ± 0.1
1
1.4 ± 0.4
1
1.5 ± 0.2
1
–
2.4 ± 0.1
1
2.8 ± 0.8
2.5 ± 0.2
D
2
O. These results are similarly consistent with two separate steps,
D
V/KNADP+
one giving rise to the primary deuterium KIE and another giving rise
D
V/KNADP+c
D
2
to the SIE. The large V/KNADP+ observed in D O is most likely an
D2O
V
2
O
2
O
2
O
2
O
2
O
2
O
D2O
equilibrium isotope effect observed as a KIE (see Discussion below).
V/KSSA
V/KNADP+
D2O
D2O
V
[2H]
Stereochemistry of hydride transfer
D2O
D2O
V/K([2H]SSA)
V/KNADP+ [2H]
1
1.6 ± 0.4
1
1
1
D
H NMR was used to probe the stereospecificity of GabD1.
V
D
D
D
2
2
2
D
+
2
2+
V/KSSA
V/KNADP+
O
O
1
Incubation of GabD1 with NADP , [ H]SSA, and Mg
led to
D
0.6 ± 0.1
0.6 ± 0.1
a
2
Experiments performed at pH 7.4 and 37 °C, in the presence of 10 mM MgCl
2
.
To determine whether the apparent SIEs observed on V/K and V with GabD1 were
O, when compared to H O,
we performed controls using 9% (w/v) glycerol, which at 25 °C has relative viscosity of
1.23 ( rel), as described elsewhere [14]. Experiments performed in 0% and 9% glycerol,
+
+
SSA was kept at 50
lM when NADP was varied, and NADP was kept at 200
lM
due to changes in viscosity due to the higher viscosity of D
2
2
when SSA was varied.
b
Value ± standard error obtained upon fitting the data to the appropriate equa-
tion. For solvent isotope effects experiments were performed in 100% H O or 85%
O.
g
2
at pH 7.4, indicate that GabD1-catalyzed reaction is not affected by the increased
viscosity to any extent (data not shown). These results indicate that the SIEs observed
are not caused by changes in viscosity associated with the increased viscosity of D O.
2
D
2
c
SSA concentration was kept at 25
lM.

9
6
L.P.S. de Carvalho et al. / Archives of Biochemistry and Biophysics 509 (2011) 90–99
2
stereospecific transfer of the deuteride from [ H]SSA to the pro-R
catalytic cysteine residue to alanine in the HsaSSADH caused great-
er sensitivity to oxidation, and abolished rescue by DTT [8]. EcoS-
+
2
1
position of NADP generating [4A- H, 4B- H] NADPH (Fig. 6). This
result indicates that GabD1 displays A-type stereospecificity.
2 2
SADH is also oxidized by H O , but this oxidation cannot be
reversed by DTT treatment. Consistent with an already maximal ef-
fect, mutation of the non-catalytic cysteine residue does not cause
any further changes in the oxidation profile of EcoSSADH [9]. From
these results, it is clear that sensitivity to oxidation of the catalytic
thiol in SSADHs is a complex phenomenon, dependent on other ac-
tive-site residues in addition to the catalytic and non-catalytic cys-
teine residues. The striking resilience of GabD1 to oxidation/
inactivation could be due to the selection pressure imposed by
the human host, Mtb’s sole reservoir, in which Mtb is constantly
challenged with reactive oxygen and nitrogen intermediates [21].
We also discovered that Mtb’s GabD1 is the first SSADH subject
Discussion
General properties of GabD1
Our studies establish GabD1 as a bona fide succinic semialde-
hyde dehydrogenase, with narrow substrate specificity for the
aldehyde substrate, as reported for SSADHs from other sources
+
+
[
4–7]. In addition, GabD1 was found to use NADP , but not NAD ,
as the preferred hydride acceptor. Our data further refute a role
for CoA as a third substrate, generating succinyl-CoA instead of
succinate. Like SSADH from A. thaliana and rat brain, GabD1 also
2
+
to activation by a divalent metal, such as Mg . Our results indicate
that GabD1 hysteresis is caused by the lack of a divalent metal acti-
vator, rather than a tightly bound inhibitor or product, like ob-
2
+
exhibits substrate inhibition by SSA [4,6]. For the Mg -activated
+
enzyme, the K
m
values for SSA and NADP are 3 and 9
l
M, respec-
values mea-
sured for A. thaliana SSADH which uses NAD as oxidant. Wheat
grain SSADH has a KmSSA value of 7.4 M and the KmNAD+ is
00 M. Rat brain SSADH also displayed a low KmSSA, 2.5 M, and
the KmNAD+ value was 30 M. Although it is clear that the high
affinity for SSA is a common trait in SSADHs, the K values for
values obtained,
range, SSA and NADP are likely the physi-
+
served for M. tuberculosis LpdA with NADP [22], E. coli MurA
tively. These values are significantly lower than the K
m
+
with UDP-N-acetylmuramic acid [23] or rabbit liver 10-formyltet-
rahydrofolate dehydrogenase with tetrahydropteroylpentagluta-
mate [24]. Some eukaryotic type 1 and type 2 ALDHs have been
l
2
l
l
2
+
reported to be activated by Mg , such as a cytosolic ALDH from
Saccharomyces cerevisiae [25], horse liver ALDH [26–28], and hu-
man liver and mitochondrial ALDH [29–31], all of which have
broad substrate specificity for the aldehyde substrates and utilize
l
m
+
NAD(P) appear to vary more. Based on the kcat/K
m
5
6
ꢀ1 ꢀ1
+
in the 10 –10 M
s
+
NAD as co-substrate. The activation of these ALDHs seems to be
ological substrates for GabD1.
similar to the activation observed with GabD1, in terms of magni-
tude of the effect. It appears that divalent cation affects very little
the Vmax of ALDHs and this is also true for GabD1. In contrast to
what was observed with type 1 and type 2 ALDHs, the effect of
Mg on GabD1 is limited to the linearization of the time course,
with only modest changes in affinity for substrates. The fact that
Interestingly, Mtb’s GabD1 is naturally resistant to oxidation by
H O . This feature distinguishes it from both HsaSSADH and EcoS-
2 2
SADH. In HsaSSADH, oxidation of the catalytic cysteine occurs, but
can be easily reversed by treatment with DTT. Mutation of the non-
2
+
2
+
2+
2+
Ca can replace Mg is intriguing, as Ca is considerably bigger
2
+
than Mg . Determination of the divalent metal binding site by
mutagenesis and crystallographic studies is essential for the fur-
ther understanding of the metal activation. However, it seems log-
ical to conclude that it is not a catalytic cofactor, as the reaction
proceeds nearly at the same rate in the absence of divalent metals.
Chemical mechanism of GabD1
The reaction catalyzed by GabD1 can be separated into three
distinct chemical steps (Scheme 2), each of which involves a cova-
lent enzyme-intermediate. In the first step, thiol/thiolate addition
to SSA generates a thiohemiacetal intermediate. Second, hydride
+
transfer to NADP generates a thioester intermediate and NADPH.
Third, hydrolysis of the thioester intermediate regenerates free en-
zyme and succinate. To characterize the chemical mechanism and
the model proposed, we conducted pH-rate and kinetic isotope ef-
fects studies.
The lack of significant pH dependence of the steady-state ki-
netic parameters of the GabD1-catalyzed reaction is quite unusual
for a dehydrogenase. That is, we observed no ionizable groups in
GabD1’s kcat pH-rate profile within the pH range of 5.5–10.0. This
result is unexpected because the reactions in question generally re-
quire (i) a general base for activation of the water molecule that
will hydrolyze the thioester intermediate, (ii) a general acid to cat-
alyze the hydride transfer step and (iii) another general base to
deprotonate the active site thiol. Ionizable groups observed in
the kcat pH-rate profile are typically important for conversion of
enzyme–substrate to enzyme–product complexes, potential iso-
merizations and product release. However, ionizable groups in-
volved with these steps will not be seen in the kcat pH-rate
profiles if binding occurs only when all ionizable groups are in
the correct protonation state when they fall outside the range ana-
lyzed, or when a conformational change is rate-limiting [15].
2
1
Fig. 6. GabD1-catalyzed production of [4A- H, 4B- H] NADPH. Spectral region of
interest of the spectra of NADPH and reaction mixtures. The assay mixture
2
2
contained, in a final volume of 0.6 mL (ca. 65% D
2
O), 1 mM [ H]SSA (ꢃ95% H),
g GabD1 in 50 mM KPi buffer pH 7.4. (a)
M NADPH, with both methylene protons, (b) spectrum of the
reaction after 25 min, (c) spectrum of the reaction after 15 min, and (d) spectrum of
+
2+
1
mM NADP , 10 mM Mg , and 100
l
Spectrum of 200
l
+
2
00
lM NADP . The chemical shift of the 4A proton is 2.76 ppm and the 4B proton is
.64 ppm. Each spectrum is the average of 128 scans.
2

L.P.S. de Carvalho et al. / Archives of Biochemistry and Biophysics 509 (2011) 90–99
97
Scheme 2.
Although unusual, a similar pH-dependence of kcat, from pH 5.5 to
normal solvent isotope effects, of different magnitude, were ob-
+
D2O
D2O
9
.5, was reported for A. suum NAD -dependent malic dehydroge-
served on
V and
V/KNADP+, indicating that a solvent-sensitive
nase-catalyzed oxidative decarboxylation of malate [32]. In the
case of GabD1, it seems rational to suggest that the carboxylate
step is partially rate-limiting for GabD1. Because these effects are
observed in both V and V/K, it is likely that they are reporting on
D2O
D2O
from Glu285 cannot be seen, as its pK
a
value is probably below
an isomerization of free enzyme. The inequality of
V and
V/
5
. The absence of a pK corresponding to the thiol from Cys319 is
a
KNADP+ indicates that the associated commitment factors are not
probably caused by an altered pK
range tested.
a
, which should be out of the
negligible. Furthermore, the linearity of our proton inventory exper-
iment denotes that a single proton is in motion during the solvent-
The lack of detectable ionizable groups on the kcat/KSSA and kcat
NADP+ versus pH plots can be more easily explained by the nature
of the residues involved in binding. The pH dependence of the kcat
K parameter reports on free substrate and on the free enzyme form
with which it combines. As can be seen in Fig. 1, GabD1 uses two
Arg residues and one Gln to bind SSA. In addition, backbone amide
nitrogen from Cys340 lies within hydrogen bonding distance of
SSA’s carbonyl oxygen, in the structure of the HsaSSADH. None of
/
sensitive step (isomerization of free enzyme). (3) When the primary
D
K
deuterium KIE is performed in D
2
O the V/KSSA decreases to unity,
/
consistent with hydride transfer and the solvent sensitive step being
localized in a different segment within the reaction. Surprisingly, an
D
inverse V/KNADP+ is now observed when the reaction is carried out
in D
2
O. Possible explanations for this result are discussed below.
2
(4) By performing SIEs using [ H]SSA we also observed a decrease
in the
D2O
D2O
V/KNADP+ value, but no change was observed on
V. De-
these residues have pK
these groups would not be identified in our analysis. SSA’s carbox-
ylate should not be seen as well, as its pK
must be ꢃ4.7, as judged
by structurally similar analogs, butanoic acid and 4-hydroxybuta-
noic acid, which have pK
is true for the kcat/KNADP+, all residues predicted to bind NADP ,
should not appear in the pH range analyzed. Therefore, the lack
of change observed in the kcat/K pH-rate profiles for GabD1 are
more likely due to the absence of ionizable groups in either binding
or rate limiting steps, within the pH range tested.
a
values in the range tested, and therefore
crease in SIE in this case is fully consistent with the idea that the
steps giving rise to the primary deuterium KIE and the SIE are differ-
ent. The decrease in the magnitude of the observed SIE when [ H]SSA
was present is considerably lower than the decrease in the magni-
tude of the primary KIEs observed when the reaction was performed
2
a
a
ꢃ 4.8 and 4.72, respectively. The same
+
in D
2
O, suggesting that the solvent-sensitive step is more rate-limit-
D2O
ing than hydride transfer. Finally, the lack of change in
V with
2
[ H]SSA indicates that the solvent sensitive step is not a chemical
step, but most likely a conformational change. Based on these results
we conclude that a conformational change of the free enzyme limits
the rate of GabD1-catalyzed reaction, while hydride transfer contrib-
utes to the steady-state rate only modestly.
Based on theory reviewed elsewhere [20,33–36], assuming lim-
iting cases for slow isotope-sensitive chemical steps and that the
rule of the geometric mean3 still applies, four conclusions can be
Inverse primary deuterium and solvent kinetic isotope effects
have been observed during multiple isotope effects experiments
with other dehydrogenases, which do not use cysteine residues
or metals for chemistry. This is the case for two other mycobacte-
rial enzymes, enoyl-ACP reductase (InhA) and 3-oxoacyl-ACP
reductase (MabA) [39,40]. Conformational changes were suggested
as potential cause for these inverse isotope effects, and conforma-
tional changes have been described to exist in both, InhA and
MabA active sites [41,42]. A similar explanation, which is fully con-
sistent with the chemistry proposed for GabD1 and the inverse pri-
mary deuterium KIE observed is offered. Thiol addition to SSA is
fast under normal conditions, while enzyme isomerization prior
to substrate binding is considerably more rate-limiting for GabD1.
Based on this sequence, it seems logical to propose that the pri-
D
drawn from our results. (1) The small but significant V/KSSA and
the unity DV obtained in H
O indicate that a step after and including
2
the first irreversible step (most likely hydrolysis of the thioester
intermediate) partially limits the overall reaction and a step from
binding of the substrate SSA up to the hydrolysis of the enzyme–thi-
oester intermediate is partially rate-limiting. Conformational
changes could lead to these slow steps, as demonstrated for malic
enzyme [37], where at pH 7 NADPH dissociation is fully rate-limit-
ing. Based on the normal isotope effect, its magnitude and chemistry
in question, it seems logical to assign it to the hydride transfer step.
This result contrasts with formate dehydrogenase, where hydride
transfer is fully rate-limiting and the primary deuterium KIE ob-
served equals the intrinsic KIE for hydride transfer [38]. (2) Large
mary deuterium KIE observed in D
the barrier to isomerization of free enzyme to its catalytic form
decrease in the steady-state turnover), which in turn, manifests
2
O is caused by an increase in
3
Analysis of multiple isotope effects becomes even more complex when two
(
hydrogens are studied because of potential deviation from the rule of geometric mean
[
34]. The rule of geometric mean states that the isotope effect in one position is
as an equilibrium isotope effect (EIE) on the thiol addition step.
An EIE is an isotope effect on the equilibrium constant of a chem-
ical step, as oppose to on its rate. In other words, the decrease in
the levels of catalytic competent form of GabD1 in D O may in-
2
crease the frequency with which the catalytic thiol attacks SSA,
as well as the reverse reaction, where the thiohemiacetal breaks
independent of the isotope effect in another position, and vice versa. Although this is
usually the case when a heavy atom is one of the isotopes varied, it may not be the
case when protons are replaced by deuterium. Currently, it is however impossible to
distinguish this, as GabD1 reaction is composed of three different chemical steps, and
analysis is further complicated by the presence of step(s) after chemistry that are
partially rate-limiting.

9
8
L.P.S. de Carvalho et al. / Archives of Biochemistry and Biophysics 509 (2011) 90–99
thiolate directly below the carboxylate from Glu306, at a distance
of 2.7 Å, which could also contribute to its activation and/or pro-
tection from oxidizing agents. Review of the literature indicates
that at least one other aldehyde dehydrogenase was shown to dis-
play this conformational change [48]. Cobessi et al. observed an
identical conformational change with the catalytic cysteine residue
+
in apo and NADP -bound structures of Streptococcus mutants
+
NADP -dependent glyceraldehyde 3-phosphate dehydrogenase
[
48]. At this stage, we cannot assign the precise role of this confor-
mational change in the mechanism of GabD1, but it must be re-
quired prior to the first chemical step. Therefore, it is tempting to
propose that the reorientation of the catalytic cysteine is the cause
of the EIE described above. By looking at the relative orientation of
the catalytic cysteine residue, after its rotation, and SSA we can
conclude that GabD1 generates a thiohemiacetal intermediate with
(S) stereochemistry (Fig. 7 and Scheme 2). The hydride from the
thiohemiacetal intermediate is then transferred to the R face of
+
NADP (pro-R position) generating the enzyme-bound thioester.
Pro-R (type A) specificity has been demonstrated for rat liver ALDH
[17], among others.
The overall chemical mechanism of GabD1 (Scheme 2) derived
from these results consists of the nucleophilic addition of the
Cys319 thiolate on the Si-face of the carbonyl of SSA generating a
Fig. 7. Conformational changed require for the initial chemical step. Superimpo-
sition of SSA (from the C340A mutant structure) on the structure of HsaSSADH,
reduced form. Cys340 thiol/thiolate is shown in two conformations, one pointing
away of the substrate (obtained in the absence of SSA) and the other, after 106°
rotation, parallel to SSA, shown in semi-transparent (modeled). This rotation would
allow the proper positioning of the thiol/thiolate to attack on the Si-face of SSA at C4
position. The distance between C4 and the thiol/thiolate is within 2.4 Å. Further-
more, the catalytic thiol/thiolate would be positioned 2.7 Å of the oxygen from
Glu306’s carboxylate, allowing its proper position and/or activation (show as
dashed black line). Image was generated using PyMol.
(
S)-thiohemiacetal intermediate. Collapse of this tetrahedral inter-
+
mediate leads to hydride transfer to the Re-face of NADP , followed
by hydrolysis of the thioester intermediate, regenerating the active
site thiolate.
Kinetic mechanism of GabD1
Although the goals of these studies were not to characterize the
kinetic mechanism of GabD1, several lines of evidence point to
non-rapid random equilibrium kinetics. First, the partial substrate
inhibition observed indicates that some degree of randomness is
present, as shown for yeast alcohol dehydrogenase [49]. Second,
the plateau observed when SSA is varied is typical of enzymes that
display non-rapid equilibrium random kinetic mechanism, where
there is a preferred path to the central complex [50,51]. This type
of mechanism has been assigned to E. coli tRNA nucleotidyltrans-
ferase [52], gentamycin acetyltransferase [53], E. coli phosphofruc-
down into free thiol and SSA. Although it would be tempting to
propose that this EIE ( V/KNADP+ = 0.6) is reporting on the hydride
D
transfer itself, EIE have been measured for hydride transfer from
+
thiohemiacetal to NAD , and these EIEs are normal and of small
magnitude [43]. Based on these observations, we interpret this in-
D
verse primary deuterium KIE in D
2
O ( V/KNADP+ = 0.6) as represent-
ing an EIE, which is caused by the ionization of the thiol happening
prior its attack on SSA (which could have a limiting value of ca.
4
0
.5). Finally, further evidence for conformational changes prior to
or linked to NADP(H) binding come from a variety of studies in other
aldehyde dehydrogenases. Marchal and Branlant demonstrated that
tokinase [54], and M. tuberculosis a-isopropylmalate synthase [55].
+
Third, the inequality of the KIEs on V and V/K values for both sub-
strates in all conditions tested strongly suggests that the forward
commitment factors are not zero, ruling out a rapid equilibrium ki-
NADP binding to glyceraldehyde 3-phosphate dehydrogenase
causes a 2.4 pK units shift in the pKapp of the catalytic cysteine,
simultaneous with changes in the protein fluorescence, indicating
D
+
netic mechanisms. Fourth, the changes observed for V/K for both
that small changes in conformation of the enzyme upon NADP bind-
2 2
substrates depending on the solvent (H O or D O) are more easily
ing exist [44]. Mitochondrial aldehyde dehydrogenase is another
+
explained by changes in the commitment factors for the substrates,
as oppose to a change in the order of addition of the substrates, in a
strictly sequential ordered mechanism. Fifth, at lower SSA concen-
good example where different bound conformations of NAD /NADH
and isomerizations of these molecules within the enzyme were
extensively characterized by X-ray crystallography, NMR and fluo-
rescence studies [45–47].
+
tration, a primary deuterium KIE is observed for NADP , indicating
D
that indeed the mechanism is random, and the lack of V/KNADP+ at
To define the stereochemical course of the reaction catalyzed by
1
higher concentrations of SSA is due to the apparent increase of the
GabD1, we employed H NMR and analysis of the structural data
+
forward commitment for NADP .
available for the human homolog. First, analysis of the structure
of the human homolog indicates that prior to thiolate addition to
SSA, the catalytic cysteine residue has to undergo a rotation around
its Cb, to allow the proper positioning of its thiolate for nucleo-
philic attack. As it can be seen on Fig. 7, in the apo structure the
Cys340’s thiolate is pointing away from SSA, blocking access and
correct positioning for nucleophilic attack. A rotation of ca. of
Together, our results suggest that GabD1 follows a non-rapid
equilibrium random kinetic mechanism with a preferred pathway
for the ternary complex, shown in Scheme S2. For GabD1, the pre-
ferred pathway most likely starts and ends with NADP+ binding
and NADPH dissociating, respectively. Isomerization of free en-
0
zyme (E ) to a form (E) that allows productive substrate binding
is also shown.
1
06° would allow its proper positioning, parallel to SSA, 2.4 Å apart
from SSA’s C4. Also, this conformation would position the Cys340’s
Acknowledgments
4
The magnitude of equilibrium deuterium isotope effects for cysteine thiols in
The authors are indebted to Dr. Carl Nathan for support and
careful reading of the manuscript. The authors acknowledge Cait-
lyn Dickinson for technical assistance, Dr. Haiteng Deng for proteo-
enzyme active sites are known to fall in the range of 0.4–0.6 (reviewed in Ref. [36]).
This represents the preference of sulfur to form S–D over S–H bonds, because of the
low stretching and bending frequencies of the S–H bond.

L.P.S. de Carvalho et al. / Archives of Biochemistry and Biophysics 509 (2011) 90–99
99
mic analysis, Drs. W.W. Cleland and Paul F. Cook for assistance
with the interpretation of isotope effects, Dr. Rafael G. da Silva
and Mrs. Clarissa M. Czekster for thoughtful discussions and criti-
cal reading of the manuscript, and Dr. Julien Vaubourgeix for prep-
aration of Figs. 1 and 7. This work was supported by NIOR
R01AI64768. The Department of Microbiology and Immunology
is supported by the William Randolph Hearst Foundation.
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