Pre-steady-state Reaction of 5-Aminolevulinate Synthase
EVIDENCE FOR A RATE-DETERMINING PRODUCT RELEASE*
Gregory A.
Hunter
§ and
Gloria C.
Ferreira
¶
**
From the
Department of Biochemistry and Molecular
Biology, College of Medicine, the ¶ Institute for Biomolecular
Science, and the
H. Lee Moffitt Cancer Center and Research
Institute, University of South Florida, Tampa, Florida 33612
 |
ABSTRACT |
5-Aminolevulinate synthase (ALAS) is the first
enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the
-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at
the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the
kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase
demonstrate that reaction with glycine plus succinyl-CoA results in a
pre-steady-state burst of quinonoid intermediate formation. Thus, a
step following binding of substrates and initial quinonoid intermediate
formation is rate-determining. The steady-state spectrum of the enzyme
is similar to that formed in the presence of 5-aminolevulinate,
suggesting that release of this product limits the overall rate.
Reaction of either glycine or 5-aminolevulinate with ALAS is slow
(kf = 0.15 s
1) and approximates
kcat. The rate constant for reaction with
glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme
that is accelerated by succinyl-CoA. The slow rate of reaction of
5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme.
Reaction of succinyl-CoA with the enzyme-glycine complex to form a
quinonoid intermediate is a biphasic process and may be irreversible.
Taken together, the data suggest that turnover is limited by release of
5-aminolevulinate or a conformational change associated with
5-aminolevulinate release.
 |
INTRODUCTION |
5-Aminolevulinate synthase
(ALAS)1 (EC 2.3.1.37)
catalyzes the condensation of glycine and succinyl-CoA to yield
coenzyme A, carbon dioxide, and ALA (1). This reaction is the first step of heme biosynthesis in non-plant eukaryotes and the
-subclass of purple bacteria (2). ALAS utilizes PLP as an obligatory cofactor and
is evolutionarily related to transaminases (3). Mammals encode two
distinct ALAS isoforms, one of which is expressed only in developing
erythrocytes (4). The erythroid-specific ALAS isoform accounts for
approximately 90% of the heme in the body, and defects in the gene are
associated with the erythropoietic disorder, X-linked sideroblastic
anemia (5).
Current understanding of the ALAS catalytic mechanism is founded
largely upon the results of radiolabeling studies conducted before the
advent of molecular cloning technology and is summarized in Fig.
1 (6-9). Following formation of an
external aldimine with glycine (II), the pro-R
proton of glycine is lost, transiently forming a quinonoid intermediate
(III) in the presence of succinyl-CoA (10). Condensation
with succinyl-CoA results in formation of an aldimine to
-amino-
-ketoadipate (V). The glycine-derived carboxyl
group is then lost (VI) and replaced by a proton to form an
aldimine to ALA (VII), which dissociates to regenerate the
holoenzyme.

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Fig. 1.
Putative ALAS reaction pathway. The
structures marked III and VI denote the two
points along the reaction pathway where the pyridoxal phosphate
cofactor functions as an electron sink, forming quinonoid
intermediates. Arrows drawn in structures II and
V indicate the movement of electrons during formation of
these putative intermediates. Structure I is drawn as the
Schiff base linkage to lysine 313 in the holoenzyme (10). Because the
protonation state of the PLP cofactor during catalysis is not known,
all structures are drawn as the protonated form of the Schiff base for
convenience only. The absolute stereochemistry of structures
IV and V about the -carbon is not known and
may be the opposite of that depicted here.
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It had been considered in the literature that the carboxyl group of
glycine might be lost before condensation with succinyl-CoA. This
possibility was discounted when it was found that in the presence of
glycine alone ALAS labilizes the pro-R proton but does not
catalyze decarboxylation. Another possible mechanism involved the
dissociation of the
-amino-
-ketoadipate intermediate (V) from the enzyme, followed by spontaneous decarboxylation to yield ALA. However, the observation that the pro-S proton
of glycine is found in the pro-S position of ALA indicates
that the decarboxylation of the
-amino-
-ketoadipate intermediate
must occur on the enzyme surface, because if it had occurred free in solution the C-5 position of product ALA would be racemic.
Whereas the chemical mechanism of ALAS is well characterized, much less
is known about the kinetic mechanism and the role the protein plays in
catalyzing the reaction. ALAS exists as a homodimer in which the active
site resides at the subunit interface (11). Steady-state kinetic
studies demonstrate a sequential mechanism with glycine binding before
succinyl-CoA and with ALA released last (10, 12). Turnover is slow,
with a kcat of 0.17 s
1 at pH 7.2 and 30 °C (13). Lysine 313 of murine erythroid ALAS forms a Schiff
base with the PLP cofactor in the absence of amino acid substrate (14).
This residue is not essential for the binding of glycine or ALA, but it
is required for catalysis (15). A crystal structure of ALAS is not
currently available, but the evolutionary relatedness to other, more
extensively characterized PLP-dependent enzymes, and in
particular aspartate aminotransferase, has provided a basis for
homology modeling studies of ALAS structure and function (13, 16, 17).
Arginine 439 has been shown to be important for the recognition and
binding of the carboxyl group of the substrate glycine (16), and
aspartate 279 has been identified as a crucial residue that enhances
the electron withdrawing capacity of the PLP cofactor by stabilizing
the protonated form of the cofactor ring nitrogen (13). Additionally,
tyrosine 121 has been implicated in binding the PLP cofactor (17). To
understand more precisely the role(s) of these and other active site
amino acids in the catalytic process, a quantitative assessment of the microscopic rate constants governing the wild-type reaction is a
necessary prerequisite.
Overproduction of recombinant murine erythroid ALAS in
Escherichia coli has allowed us to begin detailed studies of
the reaction mechanism. Here we present the results of stopped-flow
absorbance studies of the reactions of ALAS with glycine and ALA, as
well as the initial chemical event of the reaction cycle, the removal of the pro-R proton of glycine in the presence of
succinyl-CoA to form a quinonoid intermediate.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
The following reagents were from Sigma:
DEAE-Sephacel,
-mercaptoethanol, PLP, bovine serum albumin,
-ketoglutarate dehydrogenase,
-ketoglutarate, NAD+,
thiamin pyrophosphate, succinyl-CoA, ALA-hydrochloride, HEPES free
acid, and the bicinchoninic acid protein determination kit. Glycerol,
mono- and dibasic potassium phosphate, disodium EDTA dihydrate,
ammonium sulfate, magnesium chloride hexahydrate, glycine, and sodium
hydroxide were purchased from Fisher. Ultrogel AcA 44 was from IBF
Biotechnics. The bicinchoninic acid protein determination kit was from
Pierce. Sodium dodecyl sulfate-polyacrylamide electrophoresis reagents
were supplied by Bio-Rad.
Overexpression, Purification, Storage, Handling, and Analysis of
ALAS--
Recombinant murine erythroid ALAS was overproduced in
E. coli and purified as described previously (18), with the
following modifications to the protein purification procedure. 1) The
pH level of buffer A (20 mM potassium phosphate, 5 mM
-mercaptoethanol, 1 mM EDTA, 10%
glycerol) was increased to 7.5; and 2) a 20-35% (by saturation)
ammonium sulfate fractionation was utilized rather than the 0-40%
(w/v) ammonium sulfate precipitation step. Following cell disruption
and centrifugation, the magnetically stirred cytosoluble homogenate was
raised to 20% saturation with respect to ammonium sulfate by dropwise
addition of buffer A, pH 7.5, saturated with respect to ammonium
sulfate at 4 °C. The solution was centrifuged at 35,000 × g for 15 min and the supernatant brought up to 35% saturation with respect to ammonium sulfate as described above. Rapid
precipitation was typically observable when the solution reached
30-33% ammonium sulfate saturation. After a 15-min centrifugation at
35,000 × g the pellet was redissolved in a minimal
volume of buffer A, pH 7.5, and applied to the AcA 44 gel filtration
column and then to a DEAE-Sephacel column as described (18). Following purification, the enzyme was either dialyzed directly into 50 mM HEPES, pH 7.5, containing 10% v/v glycerol, or it was
concentrated by pressurized dialysis in an Amicon 8050 stir cell
equipped with a YM30 membrane before dialysis into the HEPES-glycerol
buffer. Unless otherwise noted this buffer was used in each of the
experiments reported here. The presence of glycerol was essential to
keep the enzyme solubilized. The purified ALAS protein, typically at a
concentration of 10-200 µM, was stored under liquid
nitrogen in Nalgene 2.0-ml polypropylene cryovials when not in use and thawed by incubating the cryovials in 23 °C tap water for 5-10 min.
Protein purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (19) and was never less than 95%. Enzymatic activity was determined using a continuous spectrophotometric assay
(20). Protein concentration was determined by the bicinchoninic acid
method using bovine serum albumin as standard as described previously
(13), and all protein concentrations are reported based on a subunit
molecular mass of 56,000 Da.
Stopped-flow Spectroscopy--
Rapid scanning stopped-flow
kinetic measurements were conducted using a model RSM-1000 stopped-flow
spectrophotometer equipped with a stopped-flow mixer with an optical
path length of 1.5 mm (OLIS, Inc.). The dead time of this instrument is
about 2 ms. Scans covering the wavelength range of 315-545 nm were
collected at a rate of 1000 scans/s. For reactions longer than 3 s, the collected scans were averaged to yield either 62 or 31 scans/s to condense data files to a manageable size. An external water bath was
used to maintain the temperature of the syringes (containing the
reactants) and the stopped-flow cell compartment at 30 °C. The
concentration of enzyme and ligands loaded into the syringes were
always 2-fold greater than that reported in the figure legends, such
that the reported concentrations represent the final concentrations present in the cell compartment after mixing. The concentration of
reacting ligand utilized was never less than 10-fold greater than the
enzyme concentration to ensure that pseudo-first order kinetics were
observed. Time courses at wavelengths of interest were analyzed by
fitting to equations for one to three exponentials with the SIFIT
program supplied with the instrument, where
At is the absorbance at time
t, a is the amplitude of each phase, k
is the observed rate constant for each phase, and c is the
final absorbance.
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(Eq. 1)
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Qualities of fit were assessed both from visual analysis of the
calculated residuals and from the Durbin-Watson ratio (21). Experiments
were repeated 5-8 times for each set of conditions given in Figs.
3-5. The dependences of the observed pseudo-first order rate constants
on the concentration of the reactant in excess were fit to equations
describing either a one-step (Equation 2) or two-step (Equation 3)
reaction using the nonlinear regression analysis program Enzfitter
(Biosoft Inc.).
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(Eq. 2)
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(Eq. 3)
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The resolved rate constants are reported with the standard error
of measurement obtained from the fitting.
 |
RESULTS |
Reaction of ALAS with Substrates--
The reaction of 60 µM murine erythroid ALAS with 100 mM glycine
plus 100 µM succinyl-CoA was analyzed by rapid scanning
stopped-flow spectroscopy at pH 7.5 and 30 °C (results shown in Fig.
2). Data were collected at a rate of 1000 scans/s and were averaged to yield 62 scans/s. The first event observed
is the formation of a quinonoid intermediate absorption at 510 nm. The
pre-steady-state burst of quinonoid intermediate formation reaches a
plateau at 130 ms before it decays into the steady state (Fig.
2B). Upon exhaustion of succinyl-CoA, the limiting
substrate, the steady-state quinonoid intermediate absorbance decays to
a final value slightly greater than the starting absorbance. An
expanded view of the first 3 s of the reaction is given in the
inset. This portion of the time course could be described by
a two-exponential process with rates of 12.6 and 1.82 s
1
for quinonoid intermediate formation and decay into the steady state,
respectively. The plot of the residual error associated with this fit
had some structured deviation from ideality in the early data points,
suggesting the time course might be better fit by a three-exponential
process. This was not possible for this particular time course.
however, because of an insufficient number of data points describing
the process of quinonoid intermediate formation, but it was possible
when the data were not averaged to give more data points in this region
of the time course (see below).

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Fig. 2.
Reaction of murine erythroid ALAS (60 µM) with a mixture of 100 mM glycine and
100 µM succinyl-CoA. A,
selected pre-steady-state spectra from the 2500 spectra collected during
the reaction. Spectra shown are, sequentially from the lowest to
highest absorbing at 510 nm, as follows: 16, 32, 48, 64, 80, 96, 112, and 128 ms. B, time course of the reaction at 510 nm. The
inset is an expansion of the first 3 s of the time
course. The collected data (open circles) were fit to a
two-exponential equation with rates of 12.6 and 1.83 s 1
for the fast and slow phases, respectively. The residual error
(R. E.) for this fit is also given
above the inset. C, selected spectra
from the overall time course. The spectra shown are:  , 0.000 s;
, 0.128 s; - - - , 4.000 s; and
- · · - · · -, 40.000 s.
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Representative spectra from the 2,500 spectra collected are shown in
Fig. 2C. Spectra corresponding to time 0 and 0.128 s are
identical to those given in Fig. 2A and are shown here for comparative purposes only. The spectrum at 4 s corresponds to ALAS
during steady-state catalysis and shows considerable quinonoid intermediate absorption. The spectrum at 40 s also indicates some residual quinonoid intermediate absorption because of the accumulation of ALA, which binds to ALAS some 300-fold more tightly than glycine at
pH 7.5.2
Reaction of Glycine with ALAS--
The reaction of glycine with
ALAS can be analyzed by the increase in absorbance at 425 nm (15). The
reaction of 65 µM murine erythroid ALAS with 100 mM glycine at pH 7.5 and 30 °C requires more than
30 s to reach equilibrium even at this saturating concentration of
glycine (Fig. 3). The time course was
best described by a biphasic process, with rates for the fast and slow
phases of 1.16 ± 0.04 and 0.118 ± 0.001 s
1,
respectively. The small amplitude of the fast phase resulted in a low
signal to noise ratio. It was not possible to determine reliably the
effect of glycine concentration on the observed rate constant for this
phase. It was possible, however, to determine the concentration
dependence of the slow phase, as shown in Fig. 3B. The
hyperbolic nature of the rate dependence verifies that the reaction of
glycine with ALAS is a two-step process involving a kinetically
significant intermediate. The data were fit to Equation 3 for a
two-step process. The best fit of the points gave a value of
Kd = 44 ± 9 mM for the initial
binding, followed by kf = 0.15 ± 0.01 s
1 and kr = 0.028 ± 0.006 s
1, giving an overall binding Kd of 8 mM.

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Fig. 3.
Reaction of murine erythroid ALAS (60 µM) with glycine. A,
reaction of murine erythroid ALAS with 100 mM glycine. The
time course at 425 nm is overlaid with points calculated
from the fitting to Equation 1 for a two-exponential process with
observed rate constants of 1.16 ± 0.04 and 0.118 ± 0.001 s 1. B, the rates of reaction for the slow
phase as a function of glycine concentration. The line is
the best fit curve calculated from Equation 3 with the parameter values
given under "Results." R. E., residual
error.
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Time Course of Quinonoid Intermediate Formation with ALA--
The
interaction of ALA and ALAS includes the binding and formation of a
stable quinonoid complex. The time dependence of this reaction is shown
in Fig. 4A, which records the
reaction of 2 mM ALA with 65 µM ALAS at pH
7.5 and 30 °C. No evidence of an initial increase in absorbance at
420 nm is observed prior to formation of the quinonoid intermediate,
and instead the kinetics are dominated by quinonoid complex formation.
A biphasic process describes the time course of the reaction at 510 nm,
with observed rates for the fast and slow phases of 1.43 and 0.147 s
1, respectively. The rate dependence on ALA
concentration was also determined, as shown in Fig. 4B. The
fast phase was saturable, and a fit to Equation 3 yields values of
Kd = 700 ± 300 µM,
k1 = 1.2 ± 0.2 s
1, and
k
1 = 0.6 ± 0.2 s
1,
respectively. The second, slow phase was concentration-independent, with a value of 0.15 ± 0.02 s
1.

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Fig. 4.
Pre-steady-state kinetics of reaction of
murine erythroid ALAS (60 µM) with
ALA. The data for the time course reaction of ALAS with 2 mM ALA are represented by the circles in
A. The line is from calculated points for a
two-exponential process with rates of 1.43 and 0.147 s 1.
The residual error (R. E.) for the curve fit is
given at the top. The concentration dependences of the two
phases are given in B. The fast phase of ALA dependence was
fit to Equation 3 and gave values for Kd,
kf, and kr of 700 ± 300 µM, 1.2 ± 0.2 s 1, and 0.6 ± 0.2 s 1, respectively. The slow phase of reaction with ALA was
concentration-independent with an average value of 0.15 ± 0.02 s 1 over this saturating range of ALA concentration.
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Reaction of Succinyl-CoA with the ALAS-Glycine Complex--
The
pre-steady-state reaction of 25 µM murine erythroid
ALAS-glycine complex (100 mM glycine) with 400 µM succinyl-CoA at pH 7.5 and 30 °C is depicted in
Fig. 5. A fit of the data to a biphasic
process left some structured deviation in the residuals (Fig.
5A). The Durbin-Watson ratio for this fit was less than 1, indicating a suboptimal fit. A satisfactory fit was obtained by fitting
to a triphasic process (Fig. 5B) and in the fit to the
experimental data points (Fig. 5C, solid line). The
Durbin-Watson ratio for this fit was 1.16. Binding of succinyl-CoA and
conversion of the ternary complex into a quinonoid intermediate thus
occur in two kinetic steps, the first of which can be seen at 510 nm as
a concave inflection with kobs = 53 s
1. The second, slower phase occurs with
kobs = 18 s
1 and is followed by
decay into the steady state.

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Fig. 5.
Reaction of murine erythroid ALAS-glycine
complex with succinyl-CoA. The residual errors associated with a
two-exponential fit to the data given in C are shown in
A. This fitting returned a Durbin-Watson ratio of 0.73. A
better fit was obtained by fitting the time course data to an equation
for a three-exponential process, as shown by the residuals for this fit
(B), which returned a Durbin-Watson ratio of 1.16. In
C, the time course for reaction of the ALAS-glycine complex
with 400 µM succinyl-CoA at 510 nm (circles)
is overlaid with points calculated from the fitting to
Equation 1 for a three-exponential process with observed rate constants
of 55, 18.4, and 3.16 s 1. D, rates of reaction
for the two phases of quinonoid intermediate formation as a function of succinyl-CoA concentration. The parameter values for
the fitted lines are given under "Results."
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At 25 µM ALAS-glycine, the three phases for reaction of
succinyl-CoA with the ALAS-glycine complex were resolvable, and it was
possible to determine the effect of succinyl-CoA on the observed rate
constants down to 240 µM succinyl-CoA (Fig.
5D). To acquire pseudo-first order rate constants at lower
succinyl-CoA concentrations, and thus investigate the possibility of
saturation kinetic behavior for the slow phase, the ALAS-glycine
concentration was lowered to 10 µM. At this concentration
the fast phase was not resolved from the slow phase, and a rate
constant could be computed only for the slow phase. The rate of the
fast phase was linearly dependent on the concentration of succinyl-CoA,
with a non-zero intercept, whereas the slow phase was saturable. These
data are consistent with two-step reaction kinetics of the following
type.
|
(Eq. 4)
|
k1 is defined as the slope of the best fit
line to the fast phase and equals 67,000 ± 4,000 M
1 s
1. The intercept of this
line, 29 ± 4 s
1, is equal to the sum of the other
three rate constants, k
1, k2, and k
2. A fit of
the slow phase data to Equation 3 yields values of
Kd = 110 ± 50 µM,
k2 = 21 ± 2 s
1, and
k
2 = 1.4 ± 0.5 s
1. The
maximal rate of the slow phase, equivalent to the sum of k2 and k
2, differs from
the intercept value of the fast phase by 7 ± 4 s
1,
yielding estimates of all four rate constants.
 |
DISCUSSION |
The pre-steady-state kinetics of murine erythroid ALAS have been
here investigated for the first time. The PLP cofactor at the active
site of the holoenzyme undergoes changes in absorptive properties
during catalysis that have allowed us to characterize both the
reactions of ALAS with glycine and ALA and the initial chemical event
in the forward reaction, removal of the pro-R proton of
glycine to form a quinonoid intermediate. In contrast to the quinonoid
intermediate formed in the presence of ALA, this quinonoid intermediate
is unstable and decays rapidly. The data support a model in which
release of the product ALA, or a conformational change associated with
the release of ALA, limits the turnover rate.
A pre-steady-state burst of quinonoid intermediate formation is
observed when saturating concentrations of substrates are simultaneously reacted with ALAS. The observation of burst kinetics under these conditions indicates that a step following binding of
substrates and formation of the initial quinonoid intermediate is
rate-determining. The absorbance spectrum of ALAS during steady-state catalysis is also informative, because in the steady state the largest
proportion of intermediates at any given instance will be at the
rate-determining step. The steady-state spectrum will thus be that of
the rate-limiting step if one step is completely rate-limiting, or it
will be the sum of more than one intermediate if multiple steps are
partially rate-limiting. Because considerable quinonoid intermediate is
observed during steady-state catalysis, and the formation and decay of
the first quinonoid intermediate occur faster than
kcat, this quinonoid intermediate must
correspond to the one formed in the presence of the product ALA. The
data from this experiment thus indicate that the rate-determining step is associated with product release and suggest that it is associated with the release of ALA rather than CoA or carbon dioxide.
Steady-state product inhibition studies have demonstrated that the
first step of the ALAS catalytic cycle is the binding of glycine (10,
12). In the absence of succinyl-CoA, glycine binding is a two-step
process. These steps could represent an initial noncovalent interaction
followed by slow conversion of the internal aldimine to the external
aldimine or formation of the external aldimine followed by a slow
conformational change of the enzyme, among other possibilities. Many
PLP-dependent enzymes, including aspartate aminotransferase
(22), serine hydroxymethyltransferase (23), and tryptophan synthase
(24), undergo transitions from "open" to "closed" conformations
upon amino acid binding. Differences in the two conformations have been
described in detail for aspartate aminotransferase (22, 25-28).
Closure of the enzyme around the substrate appears to function
primarily to increase substrate specificity. Interestingly,
D-amino acid aminotransferase, a PLP-dependent enzyme that does not undergo significant conformational changes during
reaction, has a broad substrate specificity (29, 30). ALAS, however,
shows strict substrate specificity for glycine; no other naturally
occurring amino acid has been found to act as a substrate (31). These
considerations argue that ALAS probably does undergo some structural
rearrangement during catalysis.
In the absence of succinyl-CoA, glycine reacts with ALAS in two kinetic
steps. We postulate that these steps represent an initial binding step
followed by a conformational change of the enzyme-glycine complex. The
second step occurs with a rate constant of 0.15 s
1, a
rate that approximates kcat, suggesting that it
may limit turnover. When 100 mM glycine and 100 µM succinyl-CoA are added to ALAS simultaneously,
however, the first observed event is formation of a quinonoid
intermediate with an observed pseudo-first order rate constant of 12 s
1. The rate constant of the second step of the reaction
of glycine with ALAS in this experiment could be no less than 12 s
1, 90-fold faster than in the absence of succinyl-CoA.
If the slow step of the glycine binding reaction is a conformational
change to a catalytically active configuration, one possible
explanation for this phenomenon is that succinyl-CoA accelerates the
rate at which this conformational change occurs. Another possibility is
that succinyl-CoA acts as an allosteric effector for glycine binding by
binding at some site distal from the active site.
Reaction of ALA with ALAS differs from reaction with glycine in that
ALA is converted to a quinonoid intermediate, whereas glycine is bound
as a stable external aldimine. ALA reacts to form a quinonoid
intermediate in two phases. The fast phase is saturable with a rapid
equilibrium Kd of 700 ± 300 µM, kf = 1.2 ± 0.2 s
1 and
kr = 0.6 ± 0.2 s
1. This is
followed by a slow phase of 0.15 ± 0.02 s
1 that is
independent of the ALA concentration and identical to the rate at which
glycine reacts in the absence of succinyl-CoA. The reaction between ALA
and ALAS thus occurs in three kinetic steps and appears to be analogous
to the reaction of glycine with ALAS with the addition of one step,
which probably represents the process of quinonoid intermediate
formation. The value of 0.6 ± 0.2 s
1 for
kr indicates that the release of ALA is at least partially rate-determining. The requirements for excess substrate, and
enough enzyme to observe sufficient signal, precluded a more precise
determination of this rate constant. If this reaction could be
monitored by fluorescence emission, the greater sensitivity of
fluorescence detection stopped flow would permit the use of lower ALAS
concentrations, which would in turn permit the measurement of
kobs at lower ALA concentrations. Fluorescence
detection might also allow a more precise determination of the reverse
rate constant for the second step of quinonoid intermediate formation,
a reaction that may be irreversible. The kinetic data obtained in
this study are summarized in Fig.
6.

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Fig. 6.
Model for the kinetic mechanism of ALAS.
The abbreviations used are: G, glycine; SCoA,
succinyl-CoA; and Q, quinonoid intermediate.
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The strict substrate specificity of ALAS for glycine is not observed
with the second substrate, succinyl-CoA (31). The maximal rates of
reaction of ALAS with glycine and a series of different CoA esters have
been reported (32). Interestingly, the maximal rates for the various
compounds are similar to succinyl-CoA and in some cases markedly
higher. Identical rates of reaction of a series of related substrates
are often considered evidence for the formation of a common structural
intermediate that precedes the rate-determining step (33-36). A
similar interpretation for ALAS is complicated by the observation that
different CoA esters would necessarily give rise to different chemical
intermediates and hence different products. A viable hypothesis would
be that the various CoA esters each stimulate the same conformational change of the enzyme to a common conformer and the breakdown of this
conformer to the initial state limits the rate of the overall reaction.
Thus, the possibility that the kinetics of ALAS catalysis are dominated
by interconversion of the enzyme between alternate conformations has
not been discounted. An oversimplified model for this would be that
ALAS exists in two conformational states, an open conformation in which
the substrates bind and a closed conformation induced by substrate
binding. Conversion of the open conformation to the closed conformation
upon binding of either glycine or ALA is slow. In the presence of both
glycine and succinyl-CoA, the rate of the conformational change is
appreciably accelerated. Because ALA alone does not accelerate the
conformational change, it appears that the impetus for lowering the
energy barrier for going to the closed conformation is supplied from
binding energy to the coenzyme A portion of succinyl-CoA. Thus, binding
interactions between ALAS and the CoA portion of succinyl-CoA may drive
a conformational change of ALAS toward the closed state wherein the
condensation between glycine and succinyl-CoA occurs. CoA is then
expelled, removing the energetic impetus for maintaining the closed
conformation, and the enzyme slowly returns to the open conformation as
ALA is released. In this model the rate-determining step is not simply the release of the product ALA from the enzyme but is a conformational change associated with release of ALA. This model could be tested in a
number of ways. Fluorescence detection stopped-flow, circular dichroism, and isotope exchange studies should each offer more insight
into the reaction mechanism.
 |
ACKNOWLEDGEMENTS |
We thank Dr. David Silverman, Dr. Chingkuang
Tu, Dr. Robert Byrne, and Alan Soli for support and advice during our
initial studies. We also thank Dr. David Silverman for critically
reading the manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant DK52053 (to G. C. F.). The rapid scanning stopped-flow
equipment was purchased with National Science Foundation Multi-User
Biological Equipment Grant DBI-9604675.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
American Heart Association, Florida Affiliate, predoctoral fellow
(Grant 9504006) and the recipient of an Institute for Biomolecular Science summer research assistantship.
**
Recipient of National Science Foundation Young Investigator Award
MCB-9257656. To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd.,
Tampa, FL 33612. Tel.: 813-974-5797; Fax: 813-974-0504; E-mail:
gferreir{at}com1.med.usf.edu.
2
G. A. Hunter and G. C. Ferreira, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
ALAS, 5-aminolevulinate synthase;
ALA, 5-aminolevulinate;
PLP, pyridoxal
5'-phosphate.
 |
REFERENCES |
-
Ahktar, M.,
and Jordan, P. M.
(1979)
in
Comprehensive Organic Chemistry: The Synthesis and Reactions of Organic Compounds (Haslam, E., ed), 1st Ed., Vol. 5, pp. 1121-44, Pergamon Press, Oxford and New York
-
Ferreira, G. C.,
and Gong, J.
(1995)
J. Bioenerg. Biomembr.
27,
151-159[Medline]
[Order article via Infotrieve]
-
Alexander, F. W.,
Sandmeier, E.,
Mehta, P. K.,
and Christen, P.
(1994)
Eur. J. Biochem.
219,
953-960[Abstract]
-
May, B. K.,
Dogra, S. C.,
Sadlon, T. J.,
Bhasker, C. R.,
Cox, T. C.,
and Bottomley, S. S.
(1995)
Prog. Nucleic Acid Res. Mol. Biol.
51,
1-51[Medline]
[Order article via Infotrieve]
-
Bottomley, S. S.,
May, B. K.,
Cox, T. C.,
Cotter, P. D.,
and Bishop, D. F.
(1995)
J. Bioenerg. Biomembr.
27,
161-168[Medline]
[Order article via Infotrieve]
-
Kikuchi, G.,
Kumar, A.,
Talmage, P.,
and Shemin, D.
(1958)
J. Biol. Chem.
233,
1214-1219[Free Full Text]
-
Zaman, Z.,
Jordan, P. M.,
and Akhtar, M.
(1973)
Biochem. J.
135,
257-263[Medline]
[Order article via Infotrieve]
-
Abboud, M. M., Jordan, P. M., and Akhtar, M. (1974)
J. Chem. Soc. Chem. Commun. 643-644
-
Laghai, A.,
and Jordan, P. M.
(1977)
Biochem. Soc. Trans.
5,
299-301[Medline]
[Order article via Infotrieve]
-
Nandi, D. L.
(1978)
J. Biol. Chem.
253,
8872-8877[Medline]
[Order article via Infotrieve]
-
Tan, D.,
and Ferreira, G. C.
(1996)
Biochemistry
35,
8934-8941[CrossRef][Medline]
[Order article via Infotrieve]
-
Fanica-Gaignier, M.,
and Clement-Metral, J.
(1973)
Eur. J. Biochem.
40,
19-24[Medline]
[Order article via Infotrieve]
-
Gong, J.,
Hunter, G. A.,
and Ferreira, G. C.
(1998)
Biochemistry
37,
3509-3517[CrossRef][Medline]
[Order article via Infotrieve]
-
Ferreira, G. C.,
Neame, P. J.,
and Dailey, H. A.
(1993)
Protein Sci.
2,
1959-1965[Abstract/Free Full Text]
-
Ferreira, G. C.,
Vajapey, U.,
Hafez, O.,
Hunter, G. A.,
and Barber, M. J.
(1995)
Protein Sci.
4,
1001-1006[Abstract/Free Full Text]
-
Tan, D.,
Harrison, T.,
Hunter, G. A.,
and Ferreira, G. C.
(1998)
Biochemistry
37,
1478-1484[CrossRef][Medline]
[Order article via Infotrieve]
-
Tan, D.,
Barber, M. J.,
and Ferreira, G. C.
(1998)
Protein Sci.
7,
1208-1213[Abstract/Free Full Text]
-
Ferreira, G. C.,
and Dailey, H. A.
(1993)
J. Biol. Chem.
268,
584-590[Abstract/Free Full Text]
-
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
-
Hunter, G. A.,
and Ferreira, G. C.
(1995)
Anal. Biochem.
226,
221-224[CrossRef][Medline]
[Order article via Infotrieve]
-
Durbin, J.,
and Watson, G. S.
(1970)
Biometrika
37,
409-414
-
Rhee, S.,
Silva, M. M.,
Hyde, C. C.,
Rogers, P. H.,
Metzler, C. M.,
Metzler, D. E.,
and Arnone, A.
(1997)
J. Biol. Chem.
272,
17293-17302[Abstract/Free Full Text]
-
Schirch, V.,
Shostak, K.,
Zamora, M.,
and Guatam-Basak, M.
(1991)
J. Biol. Chem.
266,
759-764[Abstract/Free Full Text]
-
Miles, E. W.,
Banik, U.,
Ahmed, S. A.,
Parris, K. D.,
Hyde, C. C.,
and Davies, D. R.
(1994)
in
Biochemistry of Vitamin B6 and PQQ (Marino, G., Sannia, G., and Bossa, F., eds), pp. 113-117, Birkhauser, Basel, Switzerland
-
Picot, D.,
Sandmeier, E.,
Thaller, C.,
Vincent, M. G.,
Christen, P.,
and Jansonius, J. N.
(1991)
Eur. J. Biochem.
196,
329-341[Abstract]
-
McPhalen, C. A.,
Vincent, M. G.,
Picot, D.,
Jansonius, J. N.,
Lesk, A. M.,
and Chothia, C.
(1992)
J. Mol. Biol.
227,
197-213[Medline]
[Order article via Infotrieve]
-
Jager, J.,
Moser, M.,
Sauder, U.,
and Jansonius, J. N.
(1994)
J. Mol. Biol.
239,
285-305[CrossRef][Medline]
[Order article via Infotrieve]
-
Okamoto, A.,
Higuchi, T.,
Hirotsu, K.,
Kuramitsu, S.,
and Kagamiyama, H.
(1994)
J. Biochem. (Tokyo)
116,
95-107[Abstract]
-
Peisach, D.,
Chipman, D. M.,
Van Ophem, P. W.,
Manning, J. M.,
and Ringe, D.
(1998)
Biochemistry
37,
4958-4967[CrossRef][Medline]
[Order article via Infotrieve]
-
Yonaha, K.,
Misono, H.,
Yamamoto, T.,
and Soda, K.
(1975)
J. Biol. Chem.
250,
6983-6989[Abstract]
-
Jordan, P. M.
(1990)
in
Biosynthesis of Heme and Chlorophylls (Dailey, H. A., ed), pp. 55-121, McGraw-Hill, New York
-
Shoolingin-Jordan, P. M.,
LeLean, J. E.,
and Lloyd, A. J.
(1997)
Methods Enzymol.
281,
309-316[Medline]
[Order article via Infotrieve]
-
Wang, W.,
and Hedstrom, L.
(1997)
Biochemistry
36,
8479-8483[CrossRef][Medline]
[Order article via Infotrieve]
-
Berndt, M. C.,
Bowles, M. R.,
King, G. J.,
and Zerner, B.
(1996)
Biochim. Biophys. Acta
1298,
159-166[Medline]
[Order article via Infotrieve]
-
Zhang, Z. Y.,
and Van Etten, R. L.
(1991)
Biochemistry
30,
8954-8959[Medline]
[Order article via Infotrieve]
-
Kirsch, J. F.,
and Igelstrom, M.
(1966)
Biochemistry
5,
783-791[Medline]
[Order article via Infotrieve]
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