Serine 254 enhances an induced fit mechanism in murine 5-aminolevulinate synthase

Article abstract of DOI:10.1074/jbc.M109.066548

5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3-4 Ae of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the K_m^SCoA and k_cat values of the S254A variant increased, by 25- and 2-fold, respectively, the S254T substitution decreased k_cat without altering K_m ^SCoA. Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved ～3-fold, whereas that of S254T diminished ～3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire α-oxoamine synthase family of PLP-dependent enzymes.

Full text of DOI:10.1074/jbc.M109.066548

Enzyme Catalysis and Regulation:
Serine 254 Enhances an Induced Fit
Mechanism in Murine 5-Aminolevulinate
Synthase
Thomas Lendrihas, Gregory A. Hunter and
Gloria C. Ferreira
J. Biol. Chem. 2010, 285:3351-3359.
doi: 10.1074/jbc.M109.066548 originally published online November 16, 2009
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 5, pp. 3351–3359, January 29, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Serine 254 Enhances an Induced Fit Mechanism in Murine
□
S
5-Aminolevulinate Synthase*
Received for publication, September 15, 2009, and in revised form, November 13, 2009 Published, JBC Papers in Press, November 16, 2009, DOI 10.1074/jbc.M109.066548
Thomas Lendrihas‡, Gregory A. Hunter‡, and Gloria C. Ferreira‡§¶1
From the Departments of ‡Molecular Medicine and §Chemistry and the ¶H. Lee Moffitt Cancer Center and Research Institute,
University of South Florida, Tampa, Florida 33612
5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal
5ꢀ-phosphate (PLP)-dependent enzyme, catalyzes the initial
step of heme biosynthesis in animals, fungi, and some bacteria.
Condensation of glycine and succinyl coenzyme A produces
5-aminolevulinate, coenzymeA, andcarbondioxide. X-raycrys-
tal structures of Rhodobacter capsulatus ALAS reveal that a con-
served active site serine moves to within hydrogen bonding dis-
tance of the phenolic oxygen of the PLP cofactor in the closed
encode two highly conserved but differentially expressed ALAS
genes, one of which is expressed in all tissues (ALAS1), and the
other of which is erythroid-specific (ALAS2) (2). In humans,
mutations in the ALAS2 gene can result in X-linked sideroblas-
tic anemia (3), a hypochromic and microcytic anemia charac-
terized by iron accumulation within erythroblast mitochondria
(4). Approximately one-third of X-linked sideroblastic anemia
patients are pyridoxine-responsive, and in these patients the
ALAS mutations are commonly observed in the PLP-binding
site (5, 6).
˚
substrate-bound enzyme conformation and within 3–4 A of the
thioester sulfur atom of bound succinyl-CoA. To evaluate the
role(s) of this residue in enzymatic activity, the equivalent serine
in murine erythroid ALAS was substituted with alanine or threo-
nine. Although both the KSmCoA and kcat values of the S254A
variant increased, by 25- and 2-fold, respectively, the S254T
substitution decreased kcat without altering KSmCoA. Further-
more, in relation to wild-type ALAS, the catalytic efficiency of
S254A toward glycine improved ϳ3-fold, whereas that of S254T
diminished ϳ3-fold. Circular dichroism spectroscopy revealed
that removal of the side chain hydroxyl group in the S254A var-
iant altered the microenvironment of the PLP cofactor and hin-
dered succinyl-CoA binding. Transient kinetic analyses of the
variant-catalyzed reactions and protein fluorescence quenching
upon 5-aminolevulinate binding demonstrated that the protein
conformational transition step associated with product release
was predominantly affected. We propose the following: 1) Ser-
254 is critical for formation of a competent catalytic complex by
coupling succinyl-CoA binding to enzyme conformational equi-
libria, and 2) the role of the active site serine should be extended
to the entire ␣-oxoamine synthase family of PLP-dependent
enzymes.
The ALAS chemical mechanism (Scheme 1) is complex and
involves a high degree of stereoelectronic control, with individ-
ual steps, including the following: (i) binding of glycine; (ii)
transaldimination with the active site lysine (Lys-313, murine
ALAS2 numbering) to yield an external aldimine; (iii) abstrac-
tion of the pro-R proton of glycine; (iv) condensation with
succinyl-CoA;(v)CoArelease togeneratean ␣-amino-␤-ketoadi-
pate intermediate; (vi) decarboxylation resulting in an enol-
quinonoid rapid equilibrium; (vii) protonation of the enol to
give an aldimine-bound molecule of ALA; and (viii) ultimately
release of the product (ALA) (7). This mechanistic complexity
is manifested structurally as an enzyme with an unusually high
degree of sequence conservation, as exemplified by the observa-
tion that the catalytic cores of human ALAS2 and Rhodobacter
capsulatus ALAS are 49% identical and 70% similar (8).
PLP-dependent enzymes are classified according to struc-
tural and mechanistic similarities (9). ALAS is evolutionarily
related to transaminases and is grouped within class II of the
fold type I PLP-dependent enzyme superfamily, for which
the prototypical enzyme is generally considered to be aspartate
aminotransferase (10–12). ALAS is most closely related to the
three other members of the ␣-oxoamine synthase subfamily,
each of which also catalyzes a reaction involving a small amino
acid, a CoA ester, and a 1,3-aminoketone (13, 14).
5-Aminolevulinate synthase (EC 2.3.1.37; ALAS)2 is a
homodimeric PLP-dependent enzyme that catalyzes the first
and key regulatory step of the heme biosynthetic pathway in
non-plant eukaryotes and the ␣-subclass of purple bacteria,
involving the condensation of glycine and succinyl-CoA to pro-
duce CoA, carbon dioxide, and ALA (1). Animal genomes
Aspartate aminotransferase exists in two predominant con-
formations, “open” and “closed” (10–12), and this property has
been hypothesized to be a general feature of all PLP-dependent
enzymes (15, 16). Catalysis occurs in the closed conformation,
consistent with the induced fit hypothesis, where electrostatic
and hydrophobic interactions between the substrates, cofactor,
and amino acids comprising the active site provide the ener-
getic impetus to stabilize a catalytically optimal conformation
(17).
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant DK63191 (to G. C. F.).
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental “Experimental Procedures” and an additional reference.
1 To whom correspondence should be addressed: Dept. of Molecular Medi-
cine, College of Medicine, MDC 7, University of South Florida, Tampa, FL
33612-4799. Tel.: 813-974-5797; Fax: 813-974-0504; E-mail: gferreir@
health.usf.edu.
Prior to solution of an ALAS crystal structure, kinetic data
led to the proposal that the ALAS catalytic cycle is dominated
by a rate-limiting conformational change associated with
release of ALA (7, 18–20). The crystal structures of R. capsula-
2 The abbreviations used are: ALAS, 5-aminolevulinate synthase; ALA, 5-ami-
nolevulinate; PLP, pyridoxal 5Ј-phosphate.
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Aminolevulinate Synthase-induced Fit
tus ALAS in holoenzymic and substrate-bound forms con- face closes directly over the active site channel, which extends
firmed that the enzyme adopts open and closed conformations, ϳ20 Å down into the core of the enzyme to the PLP-binding site
respectively, and allowed discrimination of the specific regions (Fig. 1) (8). A conserved threonine at the apex of the mobile
and residues of the enzyme involved in protein dynamics (8). loop forms a strong hydrogen bond (ϳ2.5 Å) with the carbox-
Although the structure in general collapses slightly around the ylate tail of succinyl-CoA in the substrate-bound structure
bound substrates, a more conformationally mobile loop of and appears to simultaneously provide molecular recogni-
amino acids located between two ␤-sheets at the subunit inter- tion for succinyl-CoA while helping to lock this substrate into
optimal stereoelectronic position
for catalysis (21). Coincident with
these changes, the side chain of
Ser-189 (R. capsulatus numbering)
migrates from noncovalently asso-
ciating with the peptide macroskel-
eton to within hydrogen bonding
distance of the PLP phenolic oxy-
gen, as well as the sulfur atom of
succinyl-CoA (8, 22, 23). These
interactions suggest that this serine
residue may be an important deter-
minant in conformer equilibrium
and catalysis by providing orienta-
tional binding energy between the
enzyme, cofactor, and substrate,
exclusively, in the closed Michaelis
complex conformation. The con-
servation of this residue in ALAS
and the other ␣-oxoamine syn-
thases further suggests an impor-
FIGURE 1. Structural models for murine erythroid ALAS based on the R. capsulatus crystal structures.
A, Michaeliscomplexmodeledbyalignmentofopenholoenzymeandclosedglycine-andsuccinyl-CoA-bound
monomeric structures. Serine 254 is hidden by the succinyl-CoA ester in this view from the perspective of the
adjacent subunit, which has been removed. The active site loop is shown in a yellow cartoon for the open and
closed conformations, and all other structural features are for the closed conformation. B, serine 254 in the
open conformation. C, serine 254 in the closed conformation with succinyl-CoA bound.
tant functionality that may be gen-
eral to these enzymes (Fig. 2). Here,
we present experiments aimed at
probing the role of this serine in
FIGURE 2. Multiple sequence alignment of phylogenetically diverse members of the ␣-oxoamine synthase family in the region of murine ALAS2
serine 254. The amino acid sequences were retrieved from public data bases (NCBI) and aligned using ClustalW (42). The conserved serine residue is
boxed and in boldface. The amino acid numbering (i.e. 254) refers to that of murine erythroid ALAS (mALAS2). Represented proteins are as follows: M. mus. AL2,
Mus musculus erythroid ALAS (156255176); H. sap. AL2, Homo sapiens erythroid ALAS (28586); H. sap. AL1, H. sapiens housekeeping ALAS (40316939); S. cer. ALA,
Saccharomyces cerevisiae ALAS (151942209); R. cap. ALA, R. capsulatus ALAS (974202); A. nig. AON, Aspergillus niger AONS (61696868); A. tha. AON, Arabidopsis
thaliana AONS (42573269); M. mar. AON, Methanococcus maripaludis AONS (1599054); E. col. AON, E. coli AONS (85674759); H. sap. KBL, H. sapiens KBL (3342906);
C. kor. KBL, Candidatus korarchaeum cryptofilum (17017433); E. col. KBL, E. coli KBL (169753078); H. sap. SPT, H. sapiens SPT (4758668); A. tha. SPT, A. thaliana SPT
(17221603); S. cer. SPT, S. cerevisiae SPT (706828), E. col. SPT, E. coli SPT (170517920). AONS, 8-amino-7-oxononanoate synthase; SPT, serine palmitoyltransferase;
KBL, 2-amino-3-oxobutyrate CoA ligase.
3352 JOURNAL OF BIOLOGICAL CHEMISTRY
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Aminolevulinate Synthase-induced Fit
catalysis by murine ALAS2. We have generated and purified the nm. Emission was measured over the wavelength range 350–
positionally equivalent S254A and S254T variants and investi- 600 nm. Buffer blanks were subtracted from the spectra.
Stopped-flow Spectroscopy—All of the experiments were car-
ried out at 30 °C in 100 mM HEPES, pH 7.5, and 10% (v/v) glyc-
erol. Because of the difference in Km for succinyl-CoA between
the two variant enzymes, two different succinyl-CoA concen-
trations were used to ensure the identification of a single
enzyme-catalyzed event. For the S254A-catalyzed reaction, the
final concentrations were as follows: 120 ␮M S254A, 130 mM
glycine, and 30 ␮M succinyl-CoA. The final concentrations of
the reactants for S254T were as follows: 50 ␮M S254T, 130 mM
glycine, and 10 ␮M succinyl-CoA. Rapid scanning stopped-flow
kinetic measurements were conducted using an OLIS model
RSM-1000 stopped-flow spectrophotometer. The dead time of
this instrument is ϳ2 ms, and the observation chamber optical
path length is 4.0 mm. Scans covering the wavelength region
270–550 nm were acquired at a rate of either 62 or 31 scans/s to
condense the resulting data files to a tractable size for data
fitting analysis. An external water bath was utilized to maintain
constant temperature of the syringes and observation chamber.
Specifically, spectral data covering the 270–550 nm range were
analyzed by global fitting to a triple exponential using the SIFIT
program supplied with the stopped-flow instrument (OLIS,
Inc.) (33). The quality of fits was judged by visual analysis of the
calculated residuals in conjunction with the Durbin-Watson
statistic (34). Single turnover data were interpreted using a
three kinetic step mechanism as described by Reaction 1.
gated the effects of these mutations on the kinetic and spectro-
scopic properties of the enzyme. The results support the pos-
tulate that this serine residue couples succinyl-CoA binding to
enzyme conformational equilibria and catalysis.
EXPERIMENTAL PROCEDURES
Reagents are listed under supplemental “Experimental
Procedures.”
Mutagenesis—The pGF23 expression plasmid encoded the
full-length sequence for the mature murine ALAS2 (24). Site-
directed mutagenesis for the S254A and S254T mouse ALAS
variants was performed on the whole plasmid pGF23 using a
previously described method (25). Experimental details are
described in the supplemental “Experimental Procedures.”
Protein Purification, SDS-PAGE, Protein Determination, and
Steady-state Analysis—Recombinant murine ALAS2 and the
S254 variants were purified from DH5␣ Escherichia coli bacterial
cells containing the overexpressed protein as described previously
(24). Purity was assessed by SDS-PAGE (26) and protein concen-
tration determined by the bicinchoninic acid method using bovine
serum albumin as the standard (27). All protein concentrations are
reported on the basis of a subunit molecular mass of 56 kDa. Enzy-
matic activity was determined by a continuous spectrophotomet-
ric assay at 30 °C (see supplemental material) (28).
Structural Analyses—The Protein Data Base files 2BWN,
2BWO, and 2BWP, corresponding to the holoenzyme, succi-
nyl-CoA-bound, and glycine-bound R. capsulatus ALAS crystal
structures, were used as templates to model the PLP-binding
core of the murine ALAS2 (8). Hydrogen bond determinations
were accomplished using Deepview/Swiss-PdbViewer software
(29, 30).
k1obs k2obs k3obs
O¡ B O¡ C O¡ D
A
REACTION 1
The observed rate constants were determined from at least
three replicate experiments, and the reported values represent
the average and standard error of measurement for each exper-
imental condition. The forward and reverse rate constants
depicted in the kinetic mechanism (Fig. 7) were obtained by
modeling single wavelength kinetic traces at 510 nm with Kin-
TekSim (Austin, TX) kinetic simulation software (35). The
eight interior rate constants were allowed to float, althoughthe
KD values were held constant as determined separately.
Transient Kinetics of the Reaction of Glycine with the Variant
Enzymes—The reactions of the murine ALAS2 variants with
glycine were performed using the same instrument as was
described for the single turnover reactions with the enzyme-
glycine complex and succinyl-CoA. The final enzyme concen-
tration was 60 ␮M. The glycine concentration was always at
least 10-fold greater than the enzyme concentration to ensure
that pseudo-first order kinetics were observed. The treatment
of the data was performed using the fitting software supplied
with the instrument. The time courses at 420 nm were fitted to
Equation 1,
Spectroscopic Measurements—All spectroscopic measure-
ments were performed with dialyzed enzyme in 20 mM HEPES,
pH 7.5, with 10% (v/v) glycerol to remove free PLP. CD spectra
were collected using an AVIV CD spectrometer calibrated for
both wavelength maxima and signal intensity with an aqueous
solution of D-10 camphorsulfonic acid (31). CD spectra
recorded from 190 to 240 nm were analyzed by the ridge regres-
sion method using a modified version of the computer program
CONTIN developed by Provencher and Glo¨ckner (32). Protein
concentrations were 10 and 100 ␮M for the near-UV-visible and
far-UV CD spectra, respectively. The final concentration of
succinyl-CoA was 100 ␮M, giving a 1:1 molar ratio of enzyme to
ligand for collection of the latter spectra. At least three CD
spectra were collected per experiment and averaged, using a
0.1-cm path length cuvette with a total volume of 300 ␮l. Blank
CD spectra contained all components of the solution except
enzyme. CD spectra containing the enzyme samples were col-
lected immediately after adding the enzyme. The spectra of the
samples containing enzyme were analyzed after subtracting the
blank spectra. Co-factor fluorescence spectra were collected on
a Shimadzu RF-5301 PC spectrofluorophotometer using pro-
tein concentrations of 2 ␮M. Spectra were measured at pH 7.5,
50 mM HEPES, and 20% (v/v) glycerol. Excitation was at 331 nm,
3
i
A ϭ
t
a eϪk t ϩ c
(Eq. 1)
͸
i
n ϭ 1
and the excitation and emission slit widths were each set to 10 where At is the absorbance at time t; a is the amplitude of each
JANUARY 29, 2010•VOLUME 285•NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 3353

Aminolevulinate Synthase-induced Fit
phase; k is the observed rate constant for each phase, and c is the where S is the concentration of substrate; k1 and k are the
final absorbance. The quality associated with the fit was deter- forward and reverse rate constants; KD is the dissociϪat1ion con-
mined by the calculated residuals and from the Durbin-Watson stant, and kobs, the observed rate constant.
ratio (34). The observed rate constants were plotted against
Intrinsic Protein Fluorescence Quenching—The presteady-
increasing concentrations of glycine, and the resulting data state kinetics of the product binding reaction of ALAS and the
were fitted to a two-step reaction process represented by Reac- two serine variants were examined by measuring changes in the
tion 2. Data fitting to Equation 2 used the nonlinear regression intrinsic protein fluorescence intensity. An OLIS RSM-1000F
analysis program SigmaPlot10 (Systat, San Jose, CA),
k1obs k2obs
rapid mixing spectrofluorimeter, equipped with a high inten-
sity xenon arc lamp, was used to monitor the reaction. The
enzyme and ligand in 20 mM HEPES, pH 7.5, and 10% glycerol
were maintained at 30 °C in separate syringes prior to their
mixing in the reaction chamber. The intrinsic protein fluores-
cence, as measured with 5 ␮M enzyme, was evaluated in the
presence of increasing concentrations of the product ALA. The
excitation wavelength and the slit width were 280 and 5 mm,
respectively. The emitted light was filtered using a cutoff filter
(WG 320; 80% transmittance at 320 nm (Edmund Optics, Bar-
rington, NJ)). Typically, 500 time points were collected for vary-
ing lengths of time, and three or more experiments were aver-
aged. Each averaged data set was then fitted to Equation 3, using
the Global fitting software provided with the instrument.
A
O¡ B O¡ C
REACTION 2
k1͓S͔
k
obs ϭ
ϩ kϪ1
(Eq. 2)
KD ϩ ͓S͔
TABLE 1
Summary of steady-state kinetic parameters
Enzyme
KGmly
KSmCoA
kcat
s
k
cat/KGmly
k
cat/KSmCoA
Ϫ1
Ϫ1
Ϫ1
mM
␮M
mMϪ1ꢁs
␮MϪ1 ꢁs
Ϫ3
Ϫ2
Ϫ3
Ϫ1
Ϫ3
Ϫ2
⌬Fobs͑t͒ ϭ A1eϪk t ϩ A0
(Eq. 3)
obs
Wild type 25 Ϯ 4 1.3 Ϯ 0.9
0.14 Ϯ 0.02
5.6 ϫ 10
1.1 ϫ 10
8.4 ϫ 10
4.2 ϫ 10
S254A
S254T
18 Ϯ 2 32 Ϯ 7
0.27 Ϯ 0.01
1.5 ϫ 10
27 Ϯ 3 1.2 Ϯ 0.3 0.050 Ϯ 0.004 1.9 ϫ 10
where Fobs(t) is the observed fluorescence change (in arbitrary
FIGURE 3. Circular dichroism and fluorescence emission spectra of ALAS and the Ser-254 variants. Spectra of wild-type ALAS (O), S254A (ꢁꢁꢁ) and S254T
(- - -). A and B, ligand-free holoenzymes; C, in the presence of 100 ␮M succinyl-CoA; D, upon excitation of the cofactor at 331 nm.
3354 JOURNAL OF BIOLOGICAL CHEMISTRY
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Aminolevulinate Synthase-induced Fit
FIGURE 4. Reaction of the Ser-254 variants (60 ␮M) with increasing con-
centrations of glycine. Data were fitted to Reaction 2 for a two-exponential
process, yieldingequilibriumandrateconstantsforS254T(KD ϭ1.5Ϯ0.4mM,
k1 ϭ 0.11 Ϯ 0.01 sϪ1, and kϪ1 ϭ 0.070 Ϯ 0.004 sϪ1) (A) and S254A (KD ϭ 6.6 Ϯ
0.6 mM, k1 ϭ 0.159 Ϯ 0.04 sϪ1, and kϪ1 ϭ 0.072 Ϯ 0.001 sϪ1) (B). The fitted rate
constants for wild-type ALAS yielded an overall binding KD of 8.0 Ϯ 0.1 mM
(18).
units) at time t; kobs is the observed first order rate constant; A1
is the pre-exponential factor, and A0 is the offset. The observed
rate constants were then plotted against ligand concentration,
and the data were fitted to Equation 4 by nonlinear regression.
The rates of dissociation (koff) and association (kon) as well as
the ligand binding constants (KD) were calculated from the
asymptotic maximal observed rate, the ordinate intercept, and
the ligand concentration (x) in Equation 4.
koffx
f͑x͒ ϭ kon ϩ
(Eq. 4)
KD ϩ x
RESULTS
Kinetic Characterization of the Ser-254 Variants—The
steady-state kinetic parameters of the Ser-254 variants were
determined, and the results are summarized in Table 1. The
mutation of serine 254 to alanine resulted in a kcat ϳ2-fold
higher than that of the wild-type ALAS value. The Km value for
succinyl-CoA was increased ϳ25-fold relative to ALAS,
although the Km value for glycine was not significantly affected.
FIGURE 5. Reaction of wild-type ALAS and the Ser-254 variants (5 ␮M)
with ALA. The observed rate constants were calculated by fitting the
decrease in intrinsic protein fluorescence over time to Equation 2 for a single
exponential process. A, resolved equilibrium and rate constants for wild-type
ALAS are as follows: KD ϭ 500 Ϯ 16 ␮M, k1 ϭ 0.120 Ϯ 0.015 sϪ1, and kϪ1 ϭ
0.140 Ϯ 0.05 sϪ1. B, for the S254A variant, the constants are as follows: KD ϭ
855 Ϯ 66 ␮M, k1 ϭ 0.235 Ϯ 0.006 sϪ1, and kϪ1 ϭ 0.29 Ϯ 0.02 sϪ1. C, for the
S254Tvariant, theconstantsareasfollows:KD ϭ832Ϯ49␮M, k1 ϭ0.19Ϯ0.09
s
Ϫ1, and kϪ1 ϭ 0.057 Ϯ 0.007 sϪ1
.
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Aminolevulinate Synthase-induced Fit
results in a broader fluorescence
emission band centered around 450
nm. The changes in the fluores-
cence emission profile for the ala-
nine variant suggest that the tauto-
meric equilibrium between at least
two forms of the PLP cofactor aldi-
mine linkage with the catalytic
lysine is perturbed.
Reaction of Glycine with the Ser-
254 Variants—The reaction of 60
␮M S254 variants with increasing
amounts of glycine resulted in an
increased absorbance at 420 nm
(Fig. 4). A global fit of the data for
the reaction of S254A with glycine
SCHEME 1
The overall catalytic efficiency for succinyl-CoA decreased yielded values for the reaction rates (k1 and kϪ1) with k1 ϭ
13-fold, although the value for glycine remained virtually 0.159 Ϯ 0.04 sϪ1 and kϪ1 ϭ 0.072 Ϯ 0.001 sϪ1 (Fig. 4B) and for
unchanged as compared with ALAS values. The replacement of the dissociation constant (KD) with KD ϭ 6.6 Ϯ 0.57 mM. The
serine 254 with threonine caused a 2.8-fold decrease in the fitting of the data corresponding to the reaction of the S254T
wild-type kcat value. The Michaelis constants for both sub- variant with glycine provided values of 0.11 Ϯ 0.01 sϪ1 and of
strates were indistinguishable from those of the wild-type 0.070 Ϯ 0.004 for k1 and kϪ1, respectively, and a KD of 1.5 Ϯ 0.39
enzyme.
Spectroscopy—The orientation and average positioning of
mM (Fig. 4A).
ALA Binding Kinetics Monitored by Intrinsic Protein
the PLP cofactor relative to the conserved serine and the active Fluorescence—The observed rates of intrinsic protein fluores-
site were perturbed by the replacement of the serine with either cence quenching upon ALA binding were determined as a func-
an alanine or a threonine. Gross structural changes evidenced tion of ALA concentration. The results are presented in Fig. 5.
by changes in the ␣-helix and ␤-sheet content of proteins can be The hyperbolic nature of the binding data indicate a two-step
identified from CD spectroscopic changes in the far-UV (36). kinetic process and may be ascribed to formation of a collision
The analysis of the UV CD spectra (Fig. 3A) by CONTIN-CD complex followed by the conformational change associated
indicates that any changes in the secondary structure between with ALA binding (7). Significantly, for each of the three
wild-type ALAS and the two ALAS variants are negligible. enzymes, the resolved rate constants for the off-rate conforma-
Locally chiral substructures that include the PLP microenvi- tional change (kϪ1) coincide with the kcat values determined
ronment modulate the visible CD characteristics of the chro- through steady-state kinetics. The dissociation constants of the
mophore. Spectra for the wild-type and variant enzymes, as variants for ALA are increased by ϳ60% over that of the wild-
holo- and succinyl-CoA-bound enzymes, were collected (Fig. type enzyme.
3). The spectra for wild-type ALAS and S254T holoenzymes
Presteady-state Reaction of the Variant Enzyme-Glycine
displayed positive dichroic bands at 330 and 420 nm. However, Complexes with Succinyl-CoA—ALAS catalysis involves the
in relation to the wild-type ALAS, the S254A variant showed a sequential binding of glycine first (I and II), then succinyl-CoA
decrease in amplitude of the 330-nm band, whereas the ampli- (III and IV), leading to an enol-quinonoid equilibrium (VI–VII)
tude in the 420-nm region increased (Fig. 3B). Wild-type ALAS after the liberation of CO2 (Scheme 1) (7). To determine the
and variant enzymes responded differently to the presence of microscopic rates associated with the lifetime of the quinonoid
100 ␮M ligand (Fig. 3C). Comparison of the CD spectra for the intermediate, we monitored the time course of the ALAS-cat-
holoenzymes with succinyl-CoA showed that spectra of wild- alyzed reaction under single turnover conditions. The time
type ALAS and S254T changed in the presence of substrate. In courses of the absorbance change were best fit to a sequential,
contrast, the spectrum of succinyl-CoA-bound S254A exhib- three-step kinetic mechanism outlined by Reaction 1. Among
ited only slightly broader bands and a small decrease in the ratio all the enzymes tested, a single step assigned to formation of a
of the dichroic intensities of the two bands when compared quinonoid intermediate followed by a biphasic step of its decay
with those observed under ligand-free conditions (i.e. (I426 nm
/
were observed (Fig. 6). For each enzyme, the global fit of the
spectral data collected at 510 nm, which has been assigned to
I330 nm
)
ScoA-bound ϭ 1.24 versus (I426 nm/I330 nm) ϭ 1.54).
Specifically, for wild-type ALAS and S254T, the amplitude of the wavelength of the quinonoid intermediate absorbance max-
both dichroic bands decreased and the 330-nm peak red- imum (18, 20), is shown in Fig. 6 as a solid line overlaid with the
shifted to ϳ350 nm. The cofactor fluorescence emission spec- time course data at 510 nm (dots). The rate constants associated
tra obtained at pH 7.5 for the S254T variant enzyme with exci- with quinonoid intermediate formation (kQf) differ between the
tation at 331 nm is similar to that of the wild-type enzyme with two variants. For S254A, the value is 4.8 Ϯ 0.2 sϪ1, a rate similar
a well formed emission maximum at ϳ385 nm (Fig. 3D). A to that of the wild-type enzyme (7). However, the rate value
notable deviation in emission maximum is observed for the for the S254T variant was decreased, showing an ϳ4-fold reduc-
S254A variant. Specifically, excitation of S254A at 331 nm tion with a rate of 1.4 Ϯ 0.3 sϪ1. These data suggest that the loss
3356 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 5•JANUARY 29, 2010

Aminolevulinate Synthase-induced Fit
of the hydroxyl group at position 254 has only a modest effect
on quinonoid intermediate formation, and therefore it does not
appear to play any obvious role in active site chemistry during
catalysis. The rates assigned to the two-step quinonoid inter-
mediate decay (kQd1 and kQd2) also differ among the variants.
Both variants have initial quinonoid intermediate decay rates
that are at least 2-fold lower than that of the wild-type enzyme
(i.e. 0.8 and 0.25 sϪ1 versus 2.0 sϪ1). However, the second step of
quinonoid intermediate decay, which most closely approxi-
mates kcat for the overall reaction and was previously assigned
to a protein conformation change associated with product
release (18, 20), is 40% faster for the S254A variant, in compar-
ison with that of the wild-type enzyme. This increased value
agrees with the greater kcat that was determined from the
steady-state kinetic analysis (Fig. 6 versus Table 1). As such, the
rates for S254A observed during the lifetime of the quinonoid
intermediate (kQf and kQd1) indicate fast transformation of the
enzyme-substrate complex to the enzyme-product complex,
followed by a comparatively faster rate of product dissociation
and the return of the conformational equilibrium to the cata-
lytically favored open conformation (kQd2).
DISCUSSION
The spectroscopic and kinetic properties of the S254A and
S254T murine ALAS variants were examined and compared
with those of the wild-type enzyme in an effort to better under-
stand the role of Ser-254 in the catalytic process. Mutation of
serine to alanine removes the side chain oxygen atom and elim-
inates the possibility of hydrogen bond formation with the PLP
cofactor and succinyl-CoA. The conservative S254T mutation
adds a methyl group and is predicted to allow for hydrogen
bonding, although some steric constraints may be introduced
due to the tight packing in this region of the active site. Com-
parison of the steady-state kinetic parameters of the variants
with those of the wild-type enzyme determined under similar
conditions hints at the unusual kinetic significance of Ser-254
(Table 1). Point mutations typically result in increased Michae-
lis constants and decreased turnover numbers, but strikingly, in
the S254A variant both of these parameters were increased. The
K
m value for succinyl-CoA was increased 25-fold over that of
the wild-type enzyme, whereas the kcat value was increased
2-fold. Although the physiological significance remains to be
established, it is worth noting that mutation of this evolution-
arily conserved residue in such a metabolically important
enzyme led to enhanced values for both kcat and catalytic effi-
ciency toward glycine (kcat/KGmly).
The kcat value for murine ALAS2 has been proposed to be
defined by a conformational change of the enzyme accompany-
ing ALA release (7). This is further supported here by the stud-
ies involving quenching of intrinsic protein fluorescence upon
product binding with both wild-type ALAS and the Ser-254
variants (Fig. 5). Indeed, the protein fluorescence-associated
ALA off-rates (kϪ1) were indistinguishable from the steady-
state kcat values (Table 1). These data also indicated that the
rate-limiting step of the reaction cycle was unaltered in
the mutated enzymes. The increase in kcat value observed for the
S254A variant may be attributable to diminished stability of the
closed conformation, leading to faster reversion to the open
FIGURE 6. Reaction of wild-type ALAS- and Ser-254 variant-glycine com-
plexes with succinyl-CoA under single turnover conditions. The data for
single turnover quinonoid intermediate formation and decay reaction kinet-
ics were fitted with the SIFIT program (OLIS, Inc.). The data (F) are overlaid
with the line representing the fitted data. A, rate constants for the three-step
sequenceinthewild-typeenzymeareasfollows:6.0, 2.0, and0.075sϪ1. B, rate
constants for the three steps in S254A-catalyzed reaction are as follows: 4.8,
0.8, and 0.19 sϪ1. C, rate constants for the three steps in S254T-catalyzed
reaction are as follows: 1.4, 0.2, and 0.037 sϪ1
.
JANUARY 29, 2010•VOLUME 285•NUMBER 5
JOURNAL OF BIOLOGICAL CHEMISTRY 3357

Aminolevulinate Synthase-induced Fit
3A). Any alterations in the confor-
mational equilibria of the active site
loop, which adopts an extended
conformation and is thus not CD
active, were not apparent in these
spectra, but the visible CD spectra
of the S254A variant do diverge sub-
stantially from those of the wild-
type and the S254T variant. The
S254A holoenzyme maximum at
435 nm was blue-shifted toward
FIGURE 7. Kinetic mechanisms of the Ser-254 variant enzymes. The single turnover quinonoid formation
and decay reaction kinetics of the variant enzymes and wild-type ALAS and the reactions of the enzymes with
glycine and ALA were used to model their kinetic mechanisms. The fits for S254A (A), S254T (B), and wild-type
ALAS (C) (7) were indistinguishable from those completed based on the global fit of spectral data. E, ALAS;
G, glycine; EG, ALAS-glycine complex; SCoA, succinyl-CoA; EGSCoA, ALAS-glycine-succinyl-CoA complex; EQ,
observable quinonoid intermediate; EALA1, ALAS-ALA internal aldimine with active site loop closed; and
EALA2, ALAS-ALA internal aldimine with active site loop open.
ϳ420 nm, and the ratio of the mean
residual ellipticity at this wave-
length to the one at 330 nm was
increased (Fig. 3B). These elliptici-
ties arise from Cotton effects associ-
ated with the ketoenamine (435 nm)
conformation and product release. In this interpretation, the
large concomitant increase in the Km value for succinyl-CoA in
the S254A variant would result not only from the direct effect of
loss of hydrogen bonding to the substrate but also from the less
direct effect of impaired enzyme conformational change as trig-
gered by the substrate, which is believed to be required for
optimal binding (7, 18). The 3-fold decrease in the kcat value
without any effect on the Km value for succinyl-CoA resulting
from the more conservative S254T mutation would then arise
primarily from enhanced stability of the closed conformation.
Both mutations substantially diminished the catalytic efficiency
with succinyl-CoA (kcat/KSmCoA) but in different ways, and
although the effects of these very conservative mutations on the
steady-state kinetic parameters may appear relatively modest,
they are presumably sufficient to have metabolically harmful
effects in vivo, given the strong evolutionary selection for serine
at this position.
and substituted aldamine (330 nm) tautomers of the PLP Schiff
base and are indicative of the microenvironment surrounding
this linkage between the cofactor and the active site lysine (39–
41). Succinyl-CoA binding to the wild-type and S254T variants
induced decreases in asymmetry of the cofactor, whereas the
S254A variant was relatively unchanged under similar condi-
tions (Fig. 3C). A logical interpretation is that the decrease in
asymmetry observed for the wild-type and S254T variants arose
from partial conversion of the internal aldimine to free PLP
aldehyde bound at the active site, as was observed in three of
four R. capsulatus crystal structure active sites upon succinyl-
CoA binding (8). In the crystal structures these events were
accompanied by transition to a closed conformation, from
which it might be concluded that the S254A variant retained
the internal aldimine in the presence of succinyl-CoA, and it
may not have been induced to adopt a closed conformation
upon binding of this substrate.
Significant effects on the cofactor microenvironment of
ALAS were induced with the S254A mutation, as determined
using fluorescence and CD spectroscopies. The differences in
the fluorescence spectra reflected alterations in cofactor tauto-
meric equilibria, which must have occurred upon loss of hydro-
gen bonding interactions between the phenolic oxygen of PLP
and the side chain of Ser-254 (Fig. 3D). We previously assigned
the 330- and 410-nm absorption species to a substituted aldam-
ine and a ketoenamine, respectively (37). The assignment of the
330-nm absorption species to a substituted aldamine, and not
the enolimine form of the Schiff base, was due to the intense
emission fluorescence of this species at 385 nm and not at the
characteristic 510-nm emission wavelength of an enolimine
(37, 38). The diminished substituted aldamine tautomer emis-
sion at 386 nm for the S254A variant is consistent with loss of
hydrogen bonding at the cofactor phenolic oxygen. An
increased proportion of the cofactor is probably in the ketoe-
namine tautomer, which we previously ascribed to the active
form of the coenzyme (37), as the 410-nm absorption species
appears to be more prominent in the S254A variant than in the
wild-type ALAS and S254T variant (data not shown). CD spec-
troscopic examination of the S254A and S254T variants in the
far-UV region confirmed that the mutations did not signifi-
Monitoring of the formation and decay of the quinonoid
reaction intermediate under single turnover conditions indi-
cated that the two Ser-254 variants follow a kinetic mechanism
similar to that of wild-type ALAS (Fig. 6, A and B). A rapid step
of ALA-bound quinonoid intermediate formation upon decar-
boxylation is followed by two successively slower decay steps,
associated with protonation of the ALA-quinonoid intermedi-
ate and ALA release, respectively (7). The quinonoid interme-
diate formation rate decreased 4-fold for the S254T variant-
catalyzed reaction, which could be explained by a change in the
flow of electrons from the site of bond scission throughout the
cofactor, precipitated by a shift in the conformational equilib-
rium toward the closed state. In the ALAS crystal structure,
PLP is noted to change position by 15° when substrate is bound
(8). Changes to the stereoelectronic relationship between the
cofactor and the ␣-carbon bonds of the external aldimine can
influence the chemical mechanism dramatically (7). Therefore,
it is possible that the hydrogen bond between serine 254 and the
phenolic oxygen of PLP may be an influential part of maintain-
ing the angle of PLP during not only formation and decay of the
quinonoid intermediate but also during the complete reaction
cycle. The increase observed in the second step of quinonoid
cantly alter the overall secondary structure of the enzymes (Fig. intermediate decay in the S254A variant is also consistent with
3358 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 5•JANUARY 29, 2010

Aminolevulinate Synthase-induced Fit
15. Jansonius, J. N., Eichele, G., Ford, G. C., Kirsch, J. F., Picot, D., Thaller, C.,
Vincent, M. G., Gehring, H., and Christen, P. (1984) Biochem. Soc. Trans.
12, 424–427
the increases in kcat and the ALA off-rate determined from
enzyme fluorescence quenching.
By utilizing the microscopic parameters obtained from the
transient kinetics for the reactions catalyzed by the variant
enzymes under single turnover conditions, coupled with the
rate constants and dissociation constants determined for the
reactions between the product or substrate with the enzymes,
we were able to model their kinetic mechanisms as shown in
Fig. 7. The simulations of the kinetic pathways revealed that the
S254T mutation significantly retards the kinetic mechanism in
relation to that of the wild-type ALAS. Conversely, the modeled
pathway for S254A highlights the increases observed in the
kinetics associated with the variant. Overall, the mechanistic
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