From the Department of Biochemistry, The Albert
Einstein College of Medicine, Bronx, New York 10461-1926 and the
§ Laboratories of Molecular Biophysics, Howard Hughes
Medical Institute, The Rockefeller University, New York, New York
10021
Received for publication, October 15, 2002, and in revised form, November 5, 2002
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ABSTRACT |
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Phosphomevalonate kinase catalyzes an
essential step in the so-called mevalonate pathway, which appears to be
the sole pathway for the biosynthesis of sterols and other isoprenoids
in mammals and archea. Despite the well documented importance of this
pathway in the cause and prevention of human disease and that it is the biosynthetic root of an enormous diverse class of metabolites, the
mechanism of phosphomevalonate kinase from any organism is not yet well
characterized. The first structure of a phosphomevalonate kinase from
Streptococcus pneumoniae was solved recently. The enzyme
exhibits an atypical P-loop that is a conserved defining feature of the
GHMP kinase superfamily. In this study, the kinetic mechanism of
the S. pneumoniae enzyme is characterized in the forward
and reverse directions using a combination of classical initial-rate
methods including alternate substrate inhibition using ADP Cholesterol, vitamin A, chlorophyll, Taxol, and geranylgeranyl and
farnesyl diphosphate (the chemical "tags" that tether proteins to
cellular membranes) represent <0.02% of the more than 30,000 known
isoprenoids (1). The members of this large complex family are
constructed in mammals, higher plants, and archea from 5-carbon building blocks donated by isopentenyl diphosphate, the end product of
the mevalonate pathway. Mevalonate is synthesized by the successive action of hydroxymethylglutaryl-CoA synthase (2) and reductase (3). It is phosphorylated by mevalonate kinase (4, 5) and then by
phosphomevalonate kinase (6, 7) to produce diphosphomevalonate, which
is decarboxylated by diphosphomevalonate decarboxylase (8) to yield the
end product, isopentenyl diphosphate. Controlling flux through this
pathway can cure human disease. The statins, a large class of
hydroxymethylglutaryl-CoA reductase inhibitors, are used widely
to regulate cholesterol biosynthesis in humans (9, 10), and altered
mevalonate kinase activity is tightly causally linked to mevalonic
aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (4,
11-13). Most bacteria have evolved an essential alternative isoprenoid
biosynthetic pathway that is a target for antibacterial therapeutics. A
notable exception is the Gram-positive cocci including
Streptococcus pneumoniae and other pathogenic
Streptococci that have retained the mevalonate pathway (14).
Despite their importance, the kinetic mechanisms of several of the
enzymes in the mevalonate pathway have yet to be determined; among
them, phosphomevalonate kinase.
Phosphomevalonate kinase (EC 2.7.4.2) catalyzes the transfer of
the The enzyme is a member of the
GHMP1 kinase superfamily (16,
17), which is composed of 13 families; eight of which have a known
catalytic function (18, 19). Superfamily members share a consensus
sequence (PX3GSSAA) that has been shown recently
to form a unique P-loop structure (7, 18, 20). Among the features that
distinguish the GHMP kinase nucleotide-binding pocket from the more
well known P-loop kinases are that the GHMP P-loop is "missing" the
catalytic lysine reside thought to stabilize negative charge that
develops at the S. The
inhibition patterns strongly support that in either direction the
substrates bind randomly to the enzyme prior to chemistry, a random
sequential bi-bi mechanism. The kinetic constants are as follows:
kcat(forward) = 3.4 s
1,
Ki(ATP) = 137 µM,
Km(ATP) = 74 µM,
Ki(pmev) = 7.7 µM,
Km(pmev) = 4.2 µM;
kcat(reverse) = 3.9 s
1,
Ki(ADP) = 410 µM,
Km(ADP) = 350 µM, Ki(ppmev) = 14 µM,
Km(ppmev) = 12 µM,
where pmev and ppmev represent
phosphomevalonate and diphosphomevalonate, respectively.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-phosphoryl group of ATP to (R)-5-phosphomevalonate, resulting in the formation of the pyrophosphoryl linkage found in
(R)-5-diphosphomevalonate (Reaction 1). Product
formation is favored slightly, Keq = 1.7 (pH = 8.0, T = 30 °C) (15). The calculated
subunit molecular mass of the S. pneumoniae enzyme used in
this study is 37,026 Da. In the crystal, the enzyme is a hexamer
organized as two back-to-back 3-fold symmetric trimers, each arranged
around a central hollow core; however, it is a monomer in solution
(7).
,
-bridging oxygen of ATP during bond cleavage
(21-23), the nucleotide is positioned relative to the P-loop such that
the adenosyl group occupies the pocket normally assigned to the
-phosphoryl acceptor, and the six-membered ring of the adenine is
rotated into a position over the ribose (i.e. syn
rather than anti).
View larger version (6K):
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Reaction 1.
Phosphomevalonate kinase was selected as a crystallization target
by the Rockefeller University contingent of the New York Structural
Genomics Research Consortium because of the lack of a representative
structure for the GHMP kinase superfamily. Targeting families without
structural representatives maximizes the impact of a structure. This
impact is extended into the area of protein function in the current
study, which uses a classical initial-rate investigation to define
fully for the first time the kinetic mechanism of a phosphomevalonate kinase.
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MATERIALS AND METHODS |
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Lactate dehydrogenase (rabbit muscle), glucose-6-phosphate
dehydrogenase (yeast), pyruvate kinase (rabbit muscle), hexokinase (yeast), -NADH, phenylmethylsulfonyl fluoride (PMSF), and pepstatin were purchased from Roche Applied Science. Nucleotides (ATP, ADP, AMP-PNP, ADP
S),
-NADP, P-enolpyruvate,
-mercaptoethanol, reduced glutathione,
polyethyleneimine-cellulose, and KH2PO4
were purchased from Sigma. Mevalonic acid was purchased from Aldrich.
NaCl, KCl, Na2HPO4, Hepes, Tris,
MgCl2, KOH, glycerol, and Luria-Bertani Miller media and
agar were purchased from Fisher.
Isopropyl-1-thio-
-D-galactopyranoside was purchased from
Labscientific, Inc. Q-Sepharose Fast Flow ion-exchange resin and GSTrap
columns were purchased from Amersham Biosciences. AG MP-1 resin was
obtained from Bio-Rad. YM-10 membranes were purchased from the
Millipore. Competent Escherichia coli BL21(DE3) were
purchased from Novagen.
Expression Plasmids-- Plasmids for the expression of mevalonate kinase and phosphomevalonate kinase from S. pneumoniae (strain R6) were kindly provided by the New York Structural Genomics Research Consortium (www.nysgrc.com). Mevalonate and phosphomevalonate kinases are expressed from a pGEX-6P-1 plasmid with a PreScission protease-cleavable N-terminal GST tag (7).
Expression and Purification of Phosphomevalonate
Kinase--
LB media (37 °C) was inoculated with freshly
transformed E. coli BL21(DE3). The cells were grown to
A600 = 0.5 and cooled to 17 °C.
Isopropyl-1-thio--D-galactopyranoside was then added to
0.70 mM, and the cells were agitated for an additional
16 h at 17 °C. The cells were harvested by centrifugation, and
the wet cell pellet was suspended in 4.2 ml/g cell paste of
phosphate-buffered saline (140 mM NaCl, 2.7 mM
KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) containing lysozyme (0.1 mg/ml), PMSF (290 µM), pepstatin A (1.5 µM), and
-mercaptoethanol (14 mM). The
cells were then disrupted by sonication at 4 °C, and cellular debris
was spun down (30 min, relative centrifugal force = 12,000 × g, 4 °C). The supernatant was loaded onto a GSTrap
column equilibrated at 4 °C with phosphate-buffered saline. The
loaded column was washed with phosphate-buffered saline, and the GST
fusion protein was eluted using Tris/Cl
(250 mM, pH 8.0), KCl (500 mM), and reduced
glutathione (10 mM). The GST tag was removed with
PreScission protease during overnight dialysis against
phosphate-buffered saline containing
-mercaptoethanol (14 mM) at 4 °C. The digest was loaded again onto the GSTrap
column. The flow-through, which contained the enzyme, was dialyzed
against Hepes/K+ (20 mM, pH 7.5), KCl (100 mM), and
-mercaptoethanol (14 mM) at
4 °C. The dialyzed protein was further purified using a Q-Sepharose Fast Flow resin (20 mM Hepes, 0-1.0 M KCl
gradient). The protein was eluted at 0.2 M KCl. Glycerol
and
-mercaptoethanol (10% v/v and 14 mM, respectively)
were then added, and the protein was frozen in aliquots and stored at
70 °C where it is stable for ~3 months.
Initial-rate Measurements-- Typically, initial rates were measured at the 16 conditions defined by a 4 × 4 matrix of substrate concentrations in which each substrate was varied in equal steps in reciprocal space from 0.20 to 5.0 × Km. The energetics of the coupling enzyme reactions are sufficiently favorable that the limiting substrate for the phosphomevalonate kinase reaction is drawn essentially completely to product in either direction. All measurements were taken during consumption of the first 7-8% of the concentration-limiting substrate. The coupling enzymes were extensively dialyzed against Hepes/K+ (50 mM, pH 7.0), and their kinetic constants were determined at the conditions used to assay phosphomevalonate kinase. The enzyme concentrations were chosen such that the coupling reactions achieved ~98% of their steady-state levels in <15 s (24).
The Forward Reaction--
ADP synthesis was coupled to the
oxidation of NADH using the enzymes pyruvate kinase and lactate
dehydrogenase (24). The formation of NAD+ was monitored by
a decrease in fluorescence (ex = 340 nm;
em = 460 nm). Controls determined that the inner filter
effect was not significant at the
6 µM (i.e.
A340
0.037) concentration of NADH used in the
experiments. The reaction progress in the inhibition studies was
monitored by the change in absorbance (340 nm), rather than
fluorescence, associated with the oxidation of NADH. Due to slight
inhibition of pyruvate kinase by AMP-PNP, the concentration of this
enzyme was increased 5-fold over the assays lacking AMP-PNP to ensure
that at the highest concentration of AMP-PNP used in the inhibition
study the coupling system reached ~98% of its steady-state levels in
<30 s. Reaction conditions were as follows: Hepes/K+ (50 mM, pH 7.0), P-enolpyruvate (1.0 mM), NADH
(0.20 mM),
-mercaptoethanol (1.0 mM), and
MgCl2 (1.0 mM + [nucleotide])
(T = 25 °C).
The Reverse Reaction--
The production of ATP was coupled to
the reduction of NADP+ using the enzymes hexokinase, and
glucose-6-phosphate dehydrogenase. NADP+ reduction was
monitored using fluorescence (ex = 340 nm;
em = 460 nm). Controls determined that the concentration
of NADPH produced during the measurements (<5 µM) was
low enough to avoid inner filter effects. To lower background
fluorescence associated with ATP contamination of ADP, it was purified
using a Q-Sepharose Fast Flow ion-exchange resin (0-1.0 M
KCl gradient). The purified ADP was desalted by loading onto an
ion-exchange resin, eluting KCL with 10 mM
TEA/HCO
-mercaptoethanol (1.0 mM), and MgCl2 (1.0 mM + [nucleotide]) (T = 25 °C).
Statistical Treatment of the Initial-rate Data--
Data were
fit with the non-linear least-squares algorithm developed by Cleland
using the models for a sequential mechanism (SEQUEN), which assumes
that the binding reactions are at equilibrium), linear competitive
inhibition (COMP), and linear noncompetitive inhibition (NONCOMP) (26).
The ADPS inhibition data were corrected for the very slight (~2-
8%) background velocity associated with turnover of the ADP
S.
The Synthesis and Purification of
Phosphomevalonate--
Phosphomevalonate was synthesized from ATP and
mevalonate using mevalonate kinase. The reaction conditions at
to were as follows: mevalonate kinase (0.20 unit/ml), ATP (4.5 mM), mevalonate (3.0 mM),
pyruvate kinase (0.5 unit/ml), P-enolpyruvate (5.0 mM),
-mercaptoethanol (1.0 mM), and MgCl2 (7.0 mM) (T = 25 °C). The reaction achieved 80% conversion of mevalonate to phosphomevalonate in 12 h.
Mevalonate kinase was purified in a manner similar to that of
phosphomevalonate kinase with the exception that the GST tag was not
cleaved away and the enzyme was not further purified by ion-exchange
chromatography. Mevalonate was prepared from mevalonic acid lactone by
incubating the lactone with three equivalents of KOH at 37 °C for
1 h (27). The solution was then adjusted to pH 7 with HCl and
diluted with water to an appropriate concentration. 13C and
1H NMR spectroscopy demonstrated that the lactone was
quantitatively converted into the free acid, and conversion back to the
lactone was not detected for at least 1 h following
neutralization. Enzymes were removed by passing the reaction mixture
through a YM-10 membrane. The solution was loaded onto an
anion-exchange resin (AG MP-1), and the compounds were eluted using a
linear 0-1.0 M KCl gradient. Phosphomevalonate eluted at
~0.35 M salt and contained <1% nucleotide, <5%
pyruvate, and <1% P-enolpyruvate. The purified phosphomevalonate was
loaded onto a 3-ml Fast-Q resin, washed with 10 mM
TEA/HCO
The Synthesis and Purification of
Diphosphomevalonate--
Diphosphomevalonate was synthesized from
mevalonate and ATP using mevalonate kinase and phosphomevalonate
kinase. Pyruvate kinase and P-enolpyruvate were added to minimize
product inhibition by ADP and to thermodynamically drive the reaction
forward. When mevalonate kinase and phosphomevalonate kinase were
simultaneously added at to, the reaction
proceeded extremely slowly. This was due to what appears to be potent
inhibition of mevalonate kinase by diphosphomevalonate. This finding
was not pursued in detail and will form the basis for further
experimentation. Inhibition was avoided by adding
phosphomevalonate kinase after the mevalonate kinase reaction had
reached completion. The reactions conditions at
to were as follows: mevalonate kinase (0.012 unit/ml), ATP (4.0 mM), mevalonate (3.0 mM),
pyruvate kinase (0.5 unit/ml), P-enolpyruvate (5.0 mM),
-mercaptoethanol (3.0 mM), and MgCl2 (7.0 mM) (T = 25 °C). Once the reaction had
reached completion (3 h, 80% conversion to product), phosphomevalonate
kinase was added to 0.07 unit/ml and the reaction was run for 12 h
at 25 °C (60% ATP was converted to diphosphomevalonate).
Purification began by removing the enzymes by passing the reaction
mixture through a YM-10 membrane. The mixture was then loaded onto an
anion-exchange resin (AG MP-1), and the compounds were eluted using a
linear KCl gradient (0-1.2 M). Diphosphomevalonate eluted
at 0.45 M salt contained <1% nucleotide and <2%
pyruvate. Diphosphomevalonate was desalted using the procedure
described above for phosphomevalonate.
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RESULTS AND DISCUSSION |
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The Forward and Reverse Reactions--
To begin to understand the
steady-state ligand-binding properties of the enzyme and to determine
its kinetic constants, a classical initial-rate study of the forward
and reverse reactions was performed. The forward and reverse reactions,
which produce ADP and ATP respectively, were monitored by following the
change in fluorescence (ex = 340 nm;
em = 460 nm) associated with either the oxidation of NADH or the reduction
of NADP+. ADP production was coupled to NADH oxidation
using the enzymes pyruvate kinase and lactate dehydrogenase, and ATP
synthesis was linked to NADP+ reduction using the enzymes
hexokinase and glucose-6-phosphate dehydrogenase (see "Materials and
Methods"). The activities of the coupling enzymes used in the assays
were determined under the conditions of the assay and were selected
such that the intrinsic lag time of the coupling systems was <15 s
(24). The energetics of the coupled reactions are sufficiently
favorable that, given the modest overall energetics for the PMK
reaction, nearly 100% of the limiting substrate for the PMK reaction
is drawn to the product in all cases. The initial rates were determined
during consumption of the first 7-8% of the limiting substrate.
The patterns of the double reciprocal plots of the initial-rate data
are consistent with a sequential mechanism in both directions (Fig.
1, A (forward) and
B (reverse)). The data were fit using the programs of
Cleland (see "Materials and Methods"), and the resulting kinetic
constants are compiled in Table I.
Intersection to the left of the 1/v axis, seen in each of
the four possible 1/v versus 1/S plots, argues
against an equilibrium-ordered mechanism (28, 29). Such mechanisms
predict intersection on the 1/v axis when the rates are
plotted versus the substrate that is added last, because the
first substrate to add is driven to saturate the enzyme at an infinite
concentration of the second due to their linked binding. The data also
argue against a Ping-Pong mechanism in which, for example, the
-phosphate is transferred first to the enzyme and then to
phosphomevalonate. Such mechanisms predict parallel lines with the
exception of the case in which the enzyme intermediate is unstable
(29). An unstable intermediate would produce ADP and be detected by the
assay; however, this was not observed. Assuming a random mechanism
(which is well supported by the inhibition studies described below),
intersection of the lines above the 1/[S] axis indicates positive
synergy in the steady-state ligand affinities. In this case, the
presence of a productively bound substrate at the active site enhances
the affinity of the enzyme for the other substrate 1.8-fold in the
forward reaction and 1.2-fold in the reverse reaction. When
kcat/Km (the pseudo-first
order rate constant governing the conversion of substrate to product at
[S]
Km) equals the rate constant for a
diffusion-limited encounter between a ligand and its active site
(~108-109 M
1
s
1) (30, 31), the encounter is rate determining and the
sum of the activation energies of all other barriers is relatively small. This is not the case for the PMK-catalyzed reaction.
kcat/Km ranges from 1.1 × 104 M
1 s
1 for
ADP to 3.3 × 106 M
1
s
1 for diphosphomevalonate. Thus, steps other than
encounter are rate determining. Using the kinetic constants in Table I,
the equilibrium constant for the overall reaction was calculated using the Haldane relationship (32) at 7.5 ± 2.2. This value is in reasonable agreement with the value of 1.8 obtained under slightly different conditions (i.e. T = 28 °C
and [MgCl2] = 12.6 mM) (15).
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The Order of Substrate Binding--
Random and ordered binding
mechanisms are distinguishable on the basis of their initial-rate
behavior toward inhibitors. Competitive dead-end inhibitors, which
typically prevent product formation by preventing the binding of one of
the substrates to the active site, are particularly useful for
determining the binding order because they result in an uncompetitive
inhibition pattern only in the ordered mechanism (34). This is the case
whether the reactant binding steps are at equilibrium or in
steady-state during turnover (34-36). When plotted in the
1/v versus 1/S format, the uncompetitive pattern
is a series of parallel lines (i.e. identical slopes) whose
1/v axis intercepts increase with an increasing concentration of inhibitor. The absence of an
[I]-dependent slope effect is readily appreciated at
[S] Km where the fraction of enzyme bound to
S in any complex can be made negligibly small by a suitable selection
of [S]. At these concentrations, I, which is incapable of binding E,
will not significantly effect [E] or the quantity of ES formed per
unit of concentration of S (i.e. the slope). In a random
mechanism, I decreases [E] regardless of [S] and thereby increases
the slope of the double reciprocal plot. Thus, the binding order is
established by determining whether an inhibitor that is competitive
against a given substrate is uncompetitive or non-competitive against
the other substrate. If uncompetitive inhibition is observed against a
given inhibitor, the mechanism is ordered and the substrate that is
competitive against that inhibitor is the second to add.
Inhibition of the Forward Reaction--
The inhibition
patterns needed to define the order of binding in the forward reaction
were obtained using the dead-end inhibitors mevalonate and AMP-PNP.
Mevalonate is competitive versus phosphomevalonate and
noncompetitive versus ATP (see Fig.
2, A and B,
respectively). Because dead-end complexes do not participate in
chemistry, their binding reactions achieve equilibrium in a
steady-state mechanism. Consequently, dead-end inhibition constants
represent dissociation constants (28, 36). The mevalonate
versus phosphomevalonate study (Fig. 2A) was
carried out at a near saturating concentration of MgATP (34 × Km; 18 × Ki). Assuming
random binding (see below) the mevalonate inhibition constant
KMev, 0.92 mM is the dissociation
constant governing the binding of mevalonate to the E·MgATP complex.
The Km of phosphomevalonate (4.2 µM),
is 220-fold less than KMev; thus, the contacts between the enzyme and phosphate play an important role in recognition. This comparison must be tempered by the fact that Km
might not equal Kd. It is notable that the addition
of the second phosphate to form diphosphomevalonate has a relatively small (3.3-fold) effect on the steady-state affinity.
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AMP-PNP is a competitive dead-end inhibitor versus ATP
(Fig. 3B).
Ki for AMP-PNP (1.8 mM) is 24 × Km for ATP. The AMP-PNP inhibition experiment was
performed under conditions ([phosphomevalonate] = 200 µM = 26 × Ki) in which
KAMP-PNP should represent the affinity of
AMP-PNP for the E-phosphomevalonate complex. The 24-fold
difference between the KAMP-PNP and the
Km for ATP suggests either that ATP binding is not
at equilibrium during turnover or that AMP-PNP does not behave like ATP
in its binding interactions with the enzyme. AMP-PNP is noncompetitive versus phosphomevalonate (Fig. 3A). Controls
established that AMP-PNP inhibition of pyruvate kinase, the coupling
enzyme used in these studies, was not significant and did not influence
the rate measurements.
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The absence of an uncompetitive inhibition pattern among these four dead-end inhibition studies argues strongly against an ordered binding mechanism and in support of a random binding scheme in the forward direction.
Inhibition of the Reverse Reaction--
The inhibition patterns
needed to determine the order of substrate binding in the reverse
direction were obtained using mevalonate, a reversible dead-end
inhibitor, and ADPS, a slow and alternate substrate. Alternate
substrates whose turnover is sufficiently slow compared with their
native counterparts that their contribution to observed velocity is
negligible, behaving like dead-end inhibitors because they effectively
stop turnover without influencing the distribution of enzyme
intermediates in the native reaction pathway. Reactions in which the
-phosphate of ATP
S is transferred to a recipient cause sulfur to
move from a diester to a monoester environment. Such reactions are
~50-fold more favorable than their ATP counterparts (37). The
enhanced stabilization is due to the sulfur-dependent
reorganization of the charge that occurs in the monoester to diester
conversion (i.e. accumulation of negative charge on sulfur
and a decrease in the P-O bond order and pKa of the
diester oxygen atoms) (38). The equilibrium constant for the
phosphomevalonate kinase-catalyzed reaction is 48-times more
favorable when ATP is replaced by ATP
S (15). It was reasoned using
the Haldane relationship that the equilibrium effect of sulfur might
manifest in the mechanism predominantly as changes in
kcat rather than Km, causing
ADP
S to be a very slow substrate and a suitable inhibitor; this was
the case. kcat for ADP
S, determined at a
near-saturating (8.8 × Km) concentration of
diphosphomevalonate (0.095 s
1 ± (0.0006)), is 41-fold
slower than kcat using ADP.
Km for ADP
S and ADP are quite comparable
(0.38 ± (0.003) and 0.41 mM, respectively) as are the
Kms for diphosphomevalonate with ADP
S and ADP
(11 ± (1.2) and 14 µM, respectively). The
considerable change in kcat and very small
changes in Km that occur when ADP is replaced by
ADP
S suggest that the binding reactions in the reverse direction may
be at equilibrium during turnover; however, this assertion assumes that
the binding interactions of ADP
S and ADP are extremely similar.
ADP
S is a non-competitive inhibitor versus
diphosphomevalonate and is competitive versus ADP (Fig.
4, A and B,
respectively).
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The kinetic constants for the R- and
S-diastereomers of ATPS in the yeast hexokinase-catalyzed
reaction are quite different (39). With Mg2+ as the
activating cation, the ratios of Km and
kcat for the R/S-isomer
pairs are 0.22 and 590, respectively. Accurate interpretation of
the results of the ADP
S experiments depends upon accurate
quantitation of the ATP
S (R and/or S) formed
during the reaction. Because of uncertainty regarding the ratio in
which the R- and S-diastereomers of ATP
S are
produced by phosphomevalonate kinase and the selective turnover of
these diastereomers is produced by hexokinase, it was important to
ensure that neither isomer was accumulating during the steady-state
measurements. For example, if the S-isomer (the poor
hexokinase substrate) was the predominant product, it might accumulate
sufficiently to cause significant error in the quantitation of both
ADP
S concentration and reaction rate. The ADP
S consumed by
phosphomevalonate kinase is regenerated by hexokinase. If regeneration
is rapid compared with consumption, the concentration of ADP
S will
remain essentially unchanged, indicating that ATP
S has not
accumulated. ADP
S is a substrate for pyruvate kinase (it produces
the S-diastereomer of ATP
S); thus, it is possible to
monitor changes in the ADP
S concentration during initial-rate
turnover of phosphomevalonate kinase using the pyruvate kinase/lactate
dehydrogenase-coupling system. The success of this method requires that
the pyruvate kinase reaction velocity be linear with ADP
S
(i.e. ADP
S < Km for the pyruvate
kinase reaction, which is 1.2 mM at the conditions used in
the ADP
S initial-rate experiments) and that the ADP
S
concentration not be depleted significantly by the pyruvate kinase
reaction. These constraints were satisfied at conditions identical to
those used in the inhibition experiments ([ADP
S] = 0.9 mM) (see legend for Fig. 4) with the exception that ADP was
not added and glucose-6-phosphate dehydrogenase was replaced by
pyruvate kinase (2.0 milliunits/ml) and lactate dehydrogenase (10 units/ml). The pyruvate kinase reaction velocity did not change
detectably (<2%) during the initial-rate stage of the
phosphomevalonate kinase reaction, indicating that the ADP
S
concentration remained essentially constant and that ATP
S did not accumulate.
Mevalonate was used as a dead-end inhibitor of the reverse reaction. It
is competitive versus diphosphomevalonate and
non-competitive versus ADP (Fig.
5, A and B,
respectively). As was the case for the forward reaction, the inhibition
patterns obtained in the four reverse reaction studies are either
competitive or noncompetitive. The lack of the uncompetitive pattern
convincingly rules out an ordered mechanism (either steady state or
equilibrium) and supports a random binding scheme.
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The data presented in this paper strongly support that the kinetic
mechanism of phosphomevalonate kinase from S. pneumoniae is
random sequential in both directions. It should be noted that this
mechanism differs from that proposed for the same enzyme from mammals.
Unlike the Streptococcal enzyme, mammalian phosphomevalonate kinase is not a member of the GHMP kinase family. Studies of the pig
liver enzyme have established that the Km for
phosphomevalonate ranges from 0.075 (27) to 0.3 mM (33),
which is 18-71-fold higher than that for the Streptococcal
enzyme. The pig liver enzyme is reportedly ordered sequential (27);
unfortunately, the data supporting this conclusion are not published.
Nevertheless, this disagreement leaves open the question of the extent
to which the mechanisms of these isoforms diverge and whether these
differences offer the opportunity for selective inhibition of this
essential enzyme, which is found in essentially all pathogenic
Gram-positive cocci (14).
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FOOTNOTES |
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* This work was supported in part by the National Institutes of Health Grants GM54469 (to T. S. L.) and P50-GM62529 (to S. K. B.) and funds from The Albert Einstein College of Medicine (to T. S. L.).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.
¶ Present address: Sunesis Pharmaceuticals, Inc., 341 Oyster Point Blvd., S. San Francisco, CA 94080.
Present address: Structural GenomiX, Inc., 10505 Roselle St.,
San Diego, CA 92121.
** To whom correspondence should be addressed: Dept. of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461-1926. Tel.: 718-430-2857; Fax: 718-430-8565; E-mail: leyh@aecom.yu.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M210551200
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ABBREVIATIONS |
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The abbreviations used are:
GHMP, galactokinase, homoserine kinase,
mevalonate kinase, phosphomevalonate kinase;
ADPS, adenosine-5'-O-(2-thiodiphosphate);
ATP
S, adenosine-5'-O-(2-thiotriphosphate);
AMP-PNP, adenosine 5'-(
,
-imino)triphosphate;
GST, glutathione
S-transferase;
TEA, triethanolamine.
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REFERENCES |
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