The Kinetic Mechanism of Phosphomevalonate Kinase*

Daniel PilloffDagger , Kristina DabovicDagger , Michael J. Romanowski§, Jeffrey B. Bonanno§, Mary DohertyDagger , Stephen K. Burley§||, and Thomas S. LeyhDagger **

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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 ADPbeta 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
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ABSTRACT
INTRODUCTION
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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 gamma -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).

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 beta ,gamma -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 gamma -phosphoryl acceptor, and the six-membered ring of the adenine is rotated into a position over the ribose (i.e. syn rather than anti).


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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.

    MATERIALS AND METHODS
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Lactate dehydrogenase (rabbit muscle), glucose-6-phosphate dehydrogenase (yeast), pyruvate kinase (rabbit muscle), hexokinase (yeast), beta -NADH, phenylmethylsulfonyl fluoride (PMSF), and pepstatin were purchased from Roche Applied Science. Nucleotides (ATP, ADP, AMP-PNP, ADPbeta S), beta -NADP, P-enolpyruvate, beta -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-beta -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-beta -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 beta -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 beta -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 beta -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 beta -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 (lambda ex = 340 nm; lambda 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), beta -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 (lambda ex = 340 nm; lambda 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<UP><SUB>3</SUB><SUP>−</SUP></UP> and then eluting ADP using 1 M TEA/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, which was removed by rotary evaporation (25). The resulting ADP contained <0.1% ATP. The conditions of the reaction were: hexokinase (1.8 units/ml) and glucose-6-phosphate dehydrogenase (0.55 units/ml), Hepes/K+ (50 mM, pH 7.0), P-enolpyruvate (1.0 mM), NADP+ (0.20 mM), beta -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 ADPbeta S inhibition data were corrected for the very slight (~2- 8%) background velocity associated with turnover of the ADPbeta 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), beta -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<UP><SUB>3</SUB><SUP>−</SUP></UP> to remove KCl, and eluted at 1.0 M TEA/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Excess TEA was removed by rotary evaporation (25).

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), beta -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.

    RESULTS AND DISCUSSION
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MATERIALS AND METHODS
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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 (lambda ex = 340 nm; lambda 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 gamma -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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Fig. 1.   Initial-rate study of the forward and reverse reactions. A, the forward reaction. Reaction progress was monitored by following the fluorescence change associated with the oxidation of NADH, which was coupled to the production of ADP using the coupling enzymes pyruvate kinase and lactate dehydrogenase. The reaction conditions were as follows: phosphomevalonate kinase (1.2 nM), ATP and phosphomevalonate (at the concentrations indicated), pyruvate kinase (1.0 unit/ml), lactate dehydrogenase (2.8 units/ml), Hepes/K+ (50 mM, pH 7.0), P-enolpyruvate (1.0 mM), NADH (5.5 µM), beta -mercaptoethanol (1.0 mM), and MgCl2 (1.0 mM + [nucleotide]) (T = 25 °C). B, the reverse reaction. Reaction progress was monitored by following the fluorescence change associated with the reduction of NADP+, which was coupled to the production of ATP using hexokinase and glucose-6-phosphate dehydrogenase. The reaction conditions were as follows: phosphomevalonate kinase (7.7 nM), ADP and diphosphomevalonate (at the concentrations indicated), hexokinase (1.8 units/ml), glucose-6-phosphate dehydrogenase (0.60 unit/ml), Hepes/K+ (50 mM, pH 7.0), glucose (10 mM), NADP+ (0.2 mM), beta -mercaptoethanol (1.0 mM), and MgCl2 (1.0 mM + [nucleotide]) (T = 25 °C). See "Materials and Methods" for further details.

                              
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Table I
Initial-rate constants for the PMK-catalyzed reaction

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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Fig. 2.   Reversible dead-end inhibition of the forward reaction. A, mevalonate versus phosphomevalonate. The conditions of the assay were as follows: phosphomevalonate kinase (2.9 nM), mevalonate and phosphomevalonate (at the concentrations indicated), ATP (2.5 mM, 34 × Km), pyruvate kinase (1.0 unit/ml), lactate dehydrogenase (2.8 units/ml), Hepes/K+ (50 mM, pH 7.0), P-enolpyruvate (1.0 mM), NADH (0.20 mM), beta -mercaptoethanol (1.0 mM), and MgCl2 (1.0 mM + [nucleotide]) (T = 25 °C). B, mevalonate versus ATP. The conditions were identical to those described in A with the exception that ATP was varied (as indicated) and phosphomevalonate was fixed at 30 µM (7.1 × Km). The lines passing through the points in A and B represent the best-fit behavior predicted by the Cleland fitting routine using the COMP and NONCOMP models, respectively. Further assay details are provided under "Materials and Methods."

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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Fig. 3.   Reversible dead-end inhibition of the forward reaction. A, AMP-PNP versus phosphomevalonate. The assay conditions were as follows: phosphomevalonate kinase (65 nM), phosphomevalonate and AMP-PNP (at the concentrations indicated), ATP was held fixed at 0.7 mM (9.6 × Km), pyruvate kinase (15 units/ml), lactate dehydrogenase (5 units/ml), Hepes/K+ (50 mM, pH 7.0), P-enolpyruvate (1.0 mM), NADH (0.20 mM), beta -mercaptoethanol (1.0 mM), and MgCl2 (1.0 mM + [nucleotide]) (T = 25 °C). B, AMP-PNP versus ATP. The conditions were identical to those described in A with the exception that AMP-PNP and ATP were varied (at the concentrations indicated) and phosphomevalonate was held fixed at 200 µM (49 × Km). Controls ensured that AMP-PNP inhibition of pyruvate kinase did not influence the rate measurements. The lines passing through the points represent the best-fit behavior predicted by the Cleland fitting programs COMP and NONCOMP. Further assay details are provided under "Materials and Methods."

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 ADPbeta S, 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 gamma -phosphate of ATPbeta 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 ATPbeta 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 ADPbeta S to be a very slow substrate and a suitable inhibitor; this was the case. kcat for ADPbeta 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 ADPbeta S and ADP are quite comparable (0.38 ± (0.003) and 0.41 mM, respectively) as are the Kms for diphosphomevalonate with ADPbeta 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 ADPbeta 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 ADPbeta S and ADP are extremely similar. ADPbeta S is a non-competitive inhibitor versus diphosphomevalonate and is competitive versus ADP (Fig. 4, A and B, respectively).


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Fig. 4.   Alternate substrate inhibition of the reverse reaction. The change in fluorescence associated with the reduction of NADP+ was used to monitor the progress of the reactions by coupling the reduction to ATP synthesis using hexokinase and glucose-6-phosphate dehydrogenase. A, ADPbeta S versus diphosphomevalonate. The reaction conditions were as follows: phosphomevalonate kinase (7.7 nM), ADPbeta S and diphosphomevalonate (at the concentrations indicated), ADP held fixed at 1.5 mM (4.3 × Km), hexokinase (1.8 units/ml), glucose-6-phosphate dehydrogenase (0.60 unit/ml), Hepes/K+ (50 mM, pH 7.0), glucose (10 mM), NADP+ (0.2 mM), beta -mercaptoethanol (1.0 mM), and MgCl2 (1.0 mM + [nucleotide]) (T = 25 °C). B, the conditions are as described in A with the exception that ADPbeta S and ADP are varied (at the concentrations indicated), and diphosphomevalonate was held fixed at 100 µM (8.6 × Km). The lines through the points represent the behavior predicted by the best fit of the data obtained from the weighted least-squares algorithm of Cleland using the COMP and NONCOMP models. For further details see "Materials and Methods."

The kinetic constants for the R- and S-diastereomers of ATPbeta S 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 ADPbeta S experiments depends upon accurate quantitation of the ATPbeta S (R and/or S) formed during the reaction. Because of uncertainty regarding the ratio in which the R- and S-diastereomers of ATPbeta 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 ADPbeta S concentration and reaction rate. The ADPbeta S consumed by phosphomevalonate kinase is regenerated by hexokinase. If regeneration is rapid compared with consumption, the concentration of ADPbeta S will remain essentially unchanged, indicating that ATPbeta S has not accumulated. ADPbeta S is a substrate for pyruvate kinase (it produces the S-diastereomer of ATPbeta S); thus, it is possible to monitor changes in the ADPbeta 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 ADPbeta S (i.e. ADPbeta S < Km for the pyruvate kinase reaction, which is 1.2 mM at the conditions used in the ADPbeta S initial-rate experiments) and that the ADPbeta 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 ([ADPbeta 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 ADPbeta S concentration remained essentially constant and that ATPbeta 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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Fig. 5.   Dead-end inhibition of the reverse reaction. The change in fluorescence associated with the reduction of NADP+ was used to monitor the progress of the reactions by coupling reduction to ATP synthesis using hexokinase and glucose-6-phosphate dehydrogenase. A, mevalonate versus diphosphomevalonate. The reaction conditions were as follows: phosphomevalonate kinase (7.7 nM), mevalonate and diphosphomevalonate (at the concentrations indicated), ADP (1.5 mM, 4.3 × Km), hexokinase (1.8 units/ml), glucose-6-phosphate dehydrogenase (0.60 unit/ml), Hepes/K+ (50 mM, pH 7.0), glucose (10 mM), NADP+ (0.2 mM), beta -mercaptoethanol (1.0 mM), and MgCl2 (1.0 mM + [nucleotide]) (T = 25 °C). B, mevalonate versus ADP. The conditions were identical to those described in A with the exception that the mevalonate and ADP are varied (at the concentrations indicated), and diphosphomevalonate was held fixed at 100 µM (8.6 × Km). The lines through the points represent the behavior predicted by the best fit of the data obtained from the weighted least-squares program of Cleland using the COMP and NONCOMP models. For further details see "Materials and Methods."

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).

    FOOTNOTES

* 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

    ABBREVIATIONS

The abbreviations used are: GHMP, galactokinase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase; ADPbeta S, adenosine-5'-O-(2-thiodiphosphate); ATPbeta S, adenosine-5'-O-(2-thiotriphosphate); AMP-PNP, adenosine 5'-(beta ,gamma -imino)triphosphate; GST, glutathione S-transferase; TEA, triethanolamine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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