Investigation of Invariant Serine/Threonine Residues in Mevalonate Kinase

TESTS OF THE FUNCTIONAL SIGNIFICANCE OF A PROPOSED SUBSTRATE BINDING MOTIF AND A SITE IMPLICATED IN HUMAN INHERITED DISEASE*

Yong-Kweon Cho, Sandra E. Ríos, Jung-Ja P. Kim, and Henry M. MiziorkoDagger

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received for publication, December 20, 2000, and in revised form, January 11, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mevalonate kinase serine/threonine residues have been implicated in substrate binding and inherited metabolic disease. Alignment of >20 mevalonate kinase sequences indicates that Ser-145, Ser-146, Ser-201, and Thr-243 are the only invariant residues with alcohol side chains. These residues have been individually mutated to alanine. Structural integrity of the mutants has been demonstrated by binding studies using fluorescent and spin-labeled ATP analogs. Kinetic characterization of the mutants indicates only modest changes in Km(ATP). Km for mevalonate increases by approx 20-fold for S146A, approx 40-fold for T243A, and 100-fold for S201A. Vmax changes for S145A, S201A, and T243A are <= 3-fold. Thus, the 65-fold activity decrease associated with the inherited human T243I mutation seems attributable to the nonconservative substitution rather than any critical catalytic function. Vmax for S146A is diminished by 4000-fold. In terms of V/KMVA, this substitution produces a 105-fold effect, suggesting an active site location and catalytic role for Ser-146. The large kcat effect suggests that Ser-146 productively orients ATP during catalysis. KD(Mg-ATP) increases by almost 40-fold for S146A, indicating a specific role for Ser-146 in liganding Mg2+-ATP. Instead of mapping within a proposed C-terminal ATP binding motif, Ser-146 is situated in a centrally located motif, which characterizes the galactokinase/homoserine kinase/ mevalonate kinase/phosphomevalonate kinase protein family. These observations represent the first functional demonstration that this region is part of the active site in these related phosphotransferases.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isoprenoid biosynthesis is accomplished by diverse pathways that use either acetyl-CoA (1) or glyceraldehyde 3-phosphate and pyruvate (2) as starting materials. Mevalonate kinase (E.C. 2.7.1.36) represents a distinctive component of the former pathway, which functions in eukaryotes, archaebacteria, and some eubacteria. In the mevalonate-mediated pathway for isoprenoid production, attention has long been focused on HMG-CoA1 reductase, which catalyzes a highly regulated step. The importance of the next reaction in this pathway, catalyzed by mevalonate kinase (Reaction 1), has become more evident based on the recent implication of this enzyme in inherited human diseases such as mevalonate kinase deficiency (Ref. 3; Mendelian inheritance in man 251170) and Dutch periodic fever/hyperimmunoglobulinemia D syndromes (Refs. 4 and 5; Mendelian inheritance in man 260920).
<UP>Mevalonate</UP>+<UP>ATP</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>M<SUP>2+</SUP></UL></LIM> mevalonate 5-phosphate+<UP>ADP</UP>

<UP><SC>Reaction</SC> 1</UP>
The diversity of species in which mevalonate kinase functions is reflected in the high degree of heterology that is apparent upon comparison of deduced sequences for this enzyme. Only 5% of the amino acids that encode the human enzyme are invariant. Included in this select group are the residues (Lys-13,2 Glu-193, Asp-204) that our laboratory has implicated previously (6, 7) in various active site functions. Inspection of mevalonate kinase's amino acid sequence does not suggest that substrate ATP binding relies on well established consensus sequences such as Walker A or Walker B motifs. However, two distinct glycine-rich stretches of amino acid sequence have been proposed as potential ATP-binding regions (8, 9), although no direct evidence that tests these hypotheses or discriminates between the two candidate sequences has appeared.

For phosphotransferase enzymes, there are numerous examples of serines or threonines that map to the active site and where the alcohol groups of the side chains support catalysis. We aligned and examined the available (>20) mevalonate kinase sequences and noted that only four residues that contain such side chains are invariant. Of these four residues, two map in one of two glycine-rich sequences proposed to be ATP-binding motifs. Additionally, the mapping of human diseases to the gene that encodes mevalonate kinase has been a prelude to identification of several missense mutations, which have been implicated as the molecular basis for the metabolic defects. An invariant threonine residue (Thr-243) falls into this category (10). On the basis of these observations, we launched an investigation of the functional significance of mevalonate kinase's invariant serine/threonine residues. The results of this study not only contribute to the mapping of the active site but also refine and expand the hypotheses that account for mevalonate kinase function and are likely to be relevant to other members of the GHMP kinase family.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

Escherichia coli strain BL21(DE3) cells and pET-3d plasmid were obtained from Novagen. DNA purification kits were purchased from Qiagen and Sigma. Mutagenic oligonucleotides were synthesized by Operon Technologies. TNP-ATP (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate) is a product of Molecular Probes. Adenosine 5'-O-[S-(acetamidoproxyl)-3-thiotriphosphate] (ATPgamma SAP) was synthesized by the method of Koteiche et al. (11). Adenosine 5'-triphosphate, beta -NADH, dithiothreitol, phosphoenolpyruvate, DL-mevalonic acid lactone, low molecular weight protein SDS-polyacrylamide gel electrophoresis markers, and any other reagents were purchased from Sigma, unless otherwise specified.

Methods

Site-directed Mutagenesis-- A polymerase chain reaction (PCR) overlap extension method (12) was used to generate the desired mutations using the following primers: S145A, 5'-GGGCTTGGGCGCCAGCGCCGC-3' and 5'-GCGGCGCTGGCGCCCAAGCCCC-3'; S146A, 5'-CTTGGGCTCCGCCGCCGCCTAC-3' and 5'-GTAGGCGGCGGCGGAGCCCAAG-3'; S201A, 5'-CGGGAACCCCGCCGGAGTG G -3' and 5'-CCACTCCGGCGGGGTTCCCG-3'; T243A, 5'-CCCTCGCAATGCCAGGGCCC-3' and 5'-GGGCCCTGGCATTGCGAGGG-3'. The DNA fragment containing the desired mutation of the human MK gene was excised from the PCR-amplified plasmid (HMK/pET-3d) with the restriction enzymes, SmaI and BglI, which cut at unique sites in this plasmid. The mutagenic fragment was isolated on an agarose gel and ligated into SmaI/BglI-digested expression plasmid that contained the remainder of the MK encoding sequence. The presence of the desired mutation and the absence of any artifactual changes in coding sequence were confirmed by automated DNA sequencing. The resulting plasmid was transformed into BL21(DE3), and mevalonate kinase was overexpressed as described by Potter and Miziorko (7).

Isolation, Assay, and Kinetic Characterization of Wild-type and Mutant Enzymes-- Human recombinant mevalonate kinase was purified as described by Potter and Miziorko (7) and stored in 20% glycerol at -80 °C. The mutants were purified following the same procedure with the exception of the hydrophobic chromatography step where the Toyopearl ether-650 column was replaced by a phenyl-agarose column. Activity was determined spectrophotometrically at 30 °C using a 1.0-ml mixture, which contained the following: HEPES, pH 7.5, 100 µmol; KCl, 100 µmol; phosphoenolpyruvate, 0.2 µmol; dithiothreitol, 0.5 µmol; NADH, 0.16 µmol; MgCl2, 10 µmol; lactate dehydrogenase, 2 units; pyruvate kinase, 4 units; ATP, 5.0 µmol; DL-mevalonate, 1.3 µmol. Activity was calculated using the extinction coefficient for NADH at 340 nm (6.22 cm-1 mM-1). Specific activity is defined as units of enzyme activity per milligram of protein, where 1 unit corresponds to product formation of 1 µmol min-1. Protein concentration was spectrophotometrically determined (A280 nm) using the extinction coefficient determined for human mevalonate kinase (epsilon 280 nm= 0.98 (mg/ml)-1). For Vmax and apparent Km experiments, measurements were performed over ATP concentrations ranging from <10-5 to >10-2 M. Depending on the mutant protein, mevalonate concentrations ranged from 12 µM to 24 mM. All reported kinetic parameters derive from nonlinear regression fits of the data using Table Curve 2D Automated Curve Fitting Software (AISN Software Inc.) and Sigmaplot (Jandel Corp.).

TNP-ATP Binding Measurements-- Measurements utilized an Aminco SLM 4800C spectrofluorimeter. 10 mM Tris-HCl buffer, pH 7.5, was used in all the experiments. For TNP-ATP titrations, the excitation wavelength was 408 nm and the emission spectra were scanned from 500 to 600 nm. For data analysis, values measured at the fluorescence emission peak of 535-540 nm (depending on the particular mutant protein) for enzyme-bound TNP-ATP were corrected for free TNP-ATP fluorescence; thus, the enhancement of fluorescence is displayed and used in all binding analyses. Sequential additions of TNP-ATP were made to a fluorescence cuvette containing approx 2.7 µM site concentration of wild-type or mutant enzyme. The relative fluorescence enhancement is plotted versus the ratio of TNP-ATP/enzyme. The binding stoichiometry for nonequilibrium complexes is determined (13-15) from the intersection point of lines fit to the low occupancy and plateau regions of the titration data by linear regression analyses (16). Calculated stoichiometries reflect binding sites per 42-kDa subunit.

ATPgamma SAP Binding Measurements-- A Varian E-109 X-band spectrometer equipped with a TE-102 cavity was used for EPR measurements. The samples were prepared such that the concentration of the enzyme was kept constant (30-40 µM) and the probe concentration was varied between 12 and 180 µM in 50 mM HEPES buffer, pH 7.5, and 100 mM KCl. The concentrations of free and bound probe were determined by comparison of the amplitude of the high field resonance line of the ATPgamma SAP spectrum (normalized for any difference in instrument gain) in the presence and absence of the protein. Under the experimental conditions employed, only unbound probe produces significant signal amplitude. The samples were loaded in 20-µl capillaries and placed in a 4-mm quartz tube; the tube was then positioned within the cavity. The spectra were recorded at the following instrument settings: microwave frequency, 9.495 GHz; microwave power, 5 milliwatts; modulation amplitude, 1 gauss; modulation frequency, 100 KHz. All samples were measured at a temperature of 23 °C. KD(Mg-ATP) values for wild-type, S145A, and S146A enzymes were measured by competitive displacement experiments (6, 7) using the same 100 mM KCl-containing buffer specified above; calculations utilize the KD(ATPgamma SAP) value determined under these conditions.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutagenic Substitution of Alcohol Side Chains-- Phosphotransferases typically use two amino acid side chains to bind cation of the M2+-ATP substrate. These are usually two acidic residues or one acidic and one serine/threonine (17). All invariant acidic residues in mevalonate kinase have been functionally evaluated by mutagenesis and kinetic characterization approaches (7); only one good candidate, namely Glu-193, emerged for assignment as cation ligand. Thus, it seemed likely that an alcohol functions as the second ligand to cation. Another potential active site role for alcohol functionalities in phosphotransferase reactions involves H-bonding to oxygens of the phosphoryl chain of ATP substrate (18-20). Elimination of the side chain in such enzymes can produce a kcat effect (21, 22) even larger than typically generated upon elimination of a cation ligand. Alignment of available deduced sequences for eukaryotic and prokaryotic mevalonate kinases indicates only four invariant residues (Ser-145, Ser-146, Ser-201, Thr-243) with alcohol groups in their side chains (Fig. 1). On the basis of the high probability that at least one of these residues is situated within the active site, a direct test of the functional importance of these amino acids seemed likely to substantially augment an active site map. To support this objective, overlap extension PCR was used to replace each of these residues with alanine. Expression of the mutant-encoding plasmids produced stable proteins, which were purified to a high degree of homogeneity; the observed yields (approx 70%) were comparable to results for wild-type enzyme. The purified mutants exhibit a single major band on SDS-polyacrylamide gel electrophoresis, migrating similarly to the 42-kDa wild-type protein subunit. The mutant proteins, like wild-type enzyme, exhibited good stability during the physical and kinetic characterization experiments described below.


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Fig. 1.   Invariant serine/threonine residues in mevalonate kinases. Deduced sequences of 6 eukaryotic proteins and 15 prokaryotic proteins were aligned using the Pileup program of the Genetics Computer Group software package. Residue numbering corresponds to the human enzyme. These sequence segments harbor the four serine/threonine residues (shown in boldface/italics) that are invariant in mevalonate kinases.

Biophysical Characterization of Mevalonate Kinase Mutants-- The tight binding fluorescent analog TNP-ATP, which incorporates a reporter group by derivatization of the ribose moiety of the nucleotide, was used to form binary complexes with wild-type and mutant MK proteins. Upon binding to MK proteins, fluorescence is highly enhanced and emission wavelength maximum "blue shifts" from 557 nm (for probe in buffer) to 535-540 nm (Fig. 2), depending upon which mevalonate kinase variant is employed to form the binary complex. This emission shift suggests a hydrophobic environment for TNP-ATP binding. The fluorescence enhancement upon titration of MK proteins with TNP-ATP is shown in Fig. 3. Attempts to fit the data to hyperbolic curves and obtain dissociation constants did not produce satisfactory results, suggesting that the binary complex exhibits nonequilibrium binding of the probe. Consequently, the low occupancy and plateau regions were independently fit to straight lines and binding stoichiometries determined from the probe/enzyme site ratio at the intersection points (13-15). Minimal differences were observed in the TNP-ATP binding stoichiometries for the various mutants (nWT = 0.64; nS145A = 0.67; nS146A = 0.48; nS201A = 0.66; nT243A = 0.76). These observations suggest that the mutants' ATP sites are comparable to that of wild-type enzyme, i.e. their active sites retain substantial structural integrity.


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Fig. 2.   Fluorescence enhancement of TNP-ATP upon binding to human mevalonate kinase. The upper trace represents the fluorescence emission spectrum (lambda max approx  540 nm) of a sample containing 1.2 µM TNP-ATP and 2.7 µM mevalonate kinase S146A in Tris-Cl (10 mM, pH 7.5). To facilitate comparison of spectral features, a sample containing an increased (approx 4-fold) concentration of TNP-ATP (4.5 µM in 10 mM Tris-Cl, pH 7.5) is used to generate the lower (base-line) spectrum (lambda max approx  557 nm). Excitation wavelength used in these experiments is 408 nm.


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Fig. 3.   Fluorescence titration of human mevalonate kinase with TNP-ATP. The solid lines represent linear regression fits of low and high occupancy data points. The stoichiometry of TNP-ATP was determined from the intersecting point of the two lines. A, wild type; B, S145A; C, S146A; D, S201A; E, T243A.

Additional characterization employed the spin-labeled ATP analog, ATPgamma SAP (11), for formation of binary complexes. Experiments were performed analogously to earlier work, which demonstrated equilibrium binding of the spin-labeled probe to wild-type enzyme (6, 7), except that 100 mM KCl was included in the sample buffer to minimize nonspecific binding. Using this probe, which contains a reporter group attached to the nucleotide's gamma -phosphoryl, binding is determined by measuring diminution of the amplitude of the signal attributable to unbound spin label. The data are amenable to Scatchard analyses (Fig. 4), which yield Kd and binding stoichiometry (n) parameters. Comparison of estimates for wild-type and mutant MK proteins (Table I) indicates that mutations do not result in substantial changes in active site binding of this probe. Wild-type MK exhibits high affinity binding (Kd = 12 µM) that is stoichiometric (n = 0.9) with respect to 42-kDa protein subunit. Mutant MK proteins exhibit Kd values that differ by <= 3-fold in comparison with wild-type Kd. The binding stoichiometries measured for the mutant MK proteins also reflect only modest differences from the estimate for wild-type enzyme. These results reinforce the conclusion that active site structure is largely unperturbed by replacement of the respective alcohol-containing residues with alanine. Thus, any substantial differences in catalytic efficiency that these mutant enzymes may exhibit can be interpreted without the concern that a structural perturbation accounts for the observations.


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Fig. 4.   Scatchard plots of EPR data for ATPgamma SAP binding to wild-type and mutant human mevalonate kinases. The concentrations of free (unbound) probe were determined by comparison of the amplitude of the high field resonance line of the ATPgamma SAP spectrum (normalized for any difference in instrument gain) in the presence and absence of the proteins. The Scatchard plots depict ATPgamma SAP binding to: wild type (A), S145A (B), S146A (C), S201A (D), and T243A (E).

                              
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Table I
ATPgamma SAP binding constants and stoichiometries for wild-type and mutant human mevalonate kinases

Kinetic Characterization of Mevalonate Kinase Mutants-- Regardless of whether alanine is substituted for Ser-145, Ser-146, Ser-201, or Thr-243, there is only a modest impact (<7-fold) on the apparent Km for ATP (Table II). In contrast, replacement of the alcohol-containing side chains has a more obvious effect on the apparent Km values for mevalonate; increases of 10-, 20-, 40-, and 100-fold are measured for S145A, S146A, T243A, and S201A, respectively. Since the S201A substitution has no major impact (<2-fold) on turnover rate, the 100-fold inflation of Km(MVA) for S201A is probably not dominated by changes in catalytic terms but could, instead, reflect a significant diminution in binding efficiency. However, in the absence of equilibrium binding constant information for wild-type and S201A proteins, such an interpretation of the inflated Km(MVA) for S201A remains provisional. As in the case of S201A, there are only minor (<3-fold) changes in Vmax for S145A and T243A. In the case of the latter mutant, no catalytic parameter changes by >20-fold and the largest effects are on Km values. These differences would be minimized under the high substrate concentrations normally employed in standard assays. This suggests that the marked depression in activity measured for the human T243I mutation (10) does not reflect any intrinsic importance of Thr-243 to catalytic efficiency, but rather the nonconservative nature of the amino acid substitution resulting from the inherited human mutation.

                              
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Table II
Apparent kinetic constants of wild-type and mutant mevalonate kinases

In view of other parameter changes that are smaller than 2 orders of magnitude, the >4000-fold diminution in Vmax observed for S146A seems noteworthy. In terms of V/KMVA, this substitution produces an effect of almost 105-fold, approaching the impact reported for elimination of the putative general base catalyst Asp-204 (7). The results certainly argue that Ser-146 maps to the active site and represent the first direct evidence to support the previous assertions that a glycine-rich sequence motif harbors part of mevalonate kinase's catalytic apparatus. Measurements of competitive displacement of ATPgamma SAP by Mg2+-ATP allow calculation (6, 7) of an equilibrium binding constant, KD(Mg-ATP). Such estimates can be more useful for evaluating the possible elimination of a cation ligand in the protein than are the binding constants for ATPgamma SAP, since this analog contains a heterocyclic substituent that contributes binding energy when localized in the hydrophobic active site of mevalonate kinase. Indeed, previous studies (7) have demonstrated that changes in KD(ATPgamma SAP) can underestimate the intrinsic importance to Mg2+-ATP binding that may be attributed to a mutated ligand. The results of these competitive displacement experiments (Table I) indicate that KD(Mg-ATP) for S146A (701 ± 14 µM) is much larger than the values measured for wild-type enzyme (19.4 ± 5.6 µM) or for S145A (32.6 ± 9.4 µM) under comparable conditions. These data reflect a selectively weakened interaction of S146A with Mg2+-ATP substrate and support assignment of S146's alcohol moiety as a ligand to divalent cation. Other possible functions for Ser-146 are discussed below in the context of the magnitude of kcat effects observed upon elimination of active site alcohol functional groups.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The detection of human gene mutations that correlate with disease has occasionally led to identification of important enzyme residues. In the case of human mevalonate kinase, the nonconservative replacement of polar Thr-243 by hydrophobic isoleucine has been documented (10). The issue concerning whether Thr-243 influences catalytic efficiency is resolved by our characterization of T243A, which does not represent as drastic a steric or polarity perturbation. The measured 2-fold diminution in Vmax indicates that the intrinsic contribution of Thr-243 to catalytic efficiency is minimal. In contrast, the T243A mutant exhibits a significant (approx 20-fold) inflation in the apparent Km(MVA), which may suggest a modest effect on mevalonic acid binding.

Although Thr-243 does not map in any sequence region previously implicated in catalysis or proposed as a consensus metabolite binding motif, the other residues investigated in this study are situated in regions that invite speculation concerning their mechanistic significance. For example, Ser-201 maps within a 12-amino acid stretch (Fig. 1) that harbors four invariant residues, including two (Glu-193 and Asp-204) previously documented (6, 7) to influence catalysis. Since the S201A substitution has little impact on Vmax, no large change in catalytic terms that contribute to Km(MVA) is expected. The inflation by 2 orders of magnitude in that parameter for S201A tempts speculation that Ser-201 is involved in mevalonate binding. Initial progress3 in elucidation of the high resolution structure of rat mevalonate kinase offers little insight into this issue, as mevalonate is not bound to the crystallized protein.

Residues 142-147 in human mevalonate kinase are highly conserved. Five invariant amino acids, including Ser-145 and Ser-146, map within this six-residue sequence. This glycine-rich, serine-containing motif appears (Fig. 5) throughout the GHMP kinase family of phosphotransferases. Moreover, isopentenyl monophosphate kinase also exhibits this motif (23). The early proposal (9) that this sequence is associated with ATP binding has not, prior to this report, been supported by experimental data. In fact, some confusion has been generated by the proposal (8) that a second glycine-rich sequence within mevalonate kinase (residues 333-357) represents an ATP binding motif. The need for two such motifs to support binding of a single nucleotide remained unclear, prompting us to attempt discrimination between these binding hypotheses. Although the S145A substitution offers surprisingly little insight into this issue, the demonstrated impact of S146's hydroxyl moiety on ATP binding as well as the large effect on mevalonate kinase's catalytic efficiency provide the first direct demonstration of the importance of the glycine-rich, serine-containing motif. These data for the glycine-rich, serine-containing motif derive additional significance upon extrapolation to other members of the GHMP kinase family of phosphotransferases. The 4000-fold impact on Vmax upon elimination of the Ser-146 hydroxyl group sharply contrasts with observations made with protein kinase A, where elimination of the ATP binding loop's serine hydroxyl has no significant effect (24). Instead, the amide backbone of the loop residue makes the important interaction with ATP.


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Fig. 5.   Homology between glycine/serine-rich loop sequences in the GHMP kinase family of proteins. Invariant residues are shown boldface and italicized. The alignment shows sequences and accession numbers for: mevalonate kinases (human, rat, Arabidopsis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Methanobacterium thermoautotrophicum, B. burgdorferi, Methanococcus jannaschii); galactokinases (Bacillus subtilis, Haemophilus influenzae, E. coli, Salmonella typhimurium, human, Candida albicans, S. cerevisiae, human 2 isozyme); homoserine kinases (E. coli, H. influenzae, M. jannaschii, B. subtilis, Streptococcus pneumoniae, Synechocystis, C. albicans, S. cerevisiae); phosphomevalonate kinases (S. cerevisiae, Enterococcus faecalis, Streptomyces sp. Cl190, Staphylococcus aureus).

Assignment of Ser-146 to the active site represents a substantial advance in defining mevalonate kinase's catalytic cavity, to which only Lys-13, Glu-193, and Asp-204 had been mapped previously (6, 7). Although this contribution is important, any demonstration that a conservative substitution has a large impact on catalysis with little negative impact on the structural integrity of the active site generates a new challenge. This involves formulation of a credible functional assignment that reconciles the available experimental data and adds insight to our understanding of how, in this particular case, the Ser-146 hydroxyl contributes to the catalytic process. For phosphotransferases, there is ample precedent to suggest that protein provides two ligands to cation of the M2+-ATP substrate (17). In the case of mevalonate kinase, our previous evaluation of acidic residues identified as invariant on the basis of a six-sequence alignment implicated two residues, Glu-193 and Asp-204, as active site residues. These residues remain invariant upon inspection of an alignment of >20 sequences currently available. As indicated in the active site schematic depicted in Fig. 6, Asp-204 has been proposed to function in general base catalysis (deprotonation of the C5 alcohol of mevalonic acid). A function in liganding to M2+-ATP was proposed for Glu-193 based, in part, on the modest kcat effects observed for the E193Q mutant. Upon substitution of an amide for this carboxyl side chain, Vmax drops by >50-fold and apparent Km values increase by approx 20-fold for ATP and approx 40-fold for mevalonate. Effects of this magnitude have been observed upon replacement of carboxyl groups that function as cation ligands in phosphofructokinase (25), 6-phosphofructo-2-kinase (22), and phosphoribulokinase (26). If the assignment of Glu-193 as cation ligand is correct, then, in the absence of any other invariant acidic residues that can be clearly be implicated in catalysis, a serine/threonine residue would be a plausible candidate for the role of second cation ligand.


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Fig. 6.   Schematic view of the active site of mevalonate kinase. Residues mapped to the active site on the basis of work presented in this or earlier reports are indicated. Possible liganding/H-bonding functions for Ser-146 are illustrated. Lys-13 has been suggested (6) to interact with ATP. Glu-193 has been proposed (7) to be a ligand to Mg2+-ATP. Asp-204 has been proposed (7) to function as a general base catalyst in deprotonation of mevalonic acid.

The results and analysis presented above implicate Ser-146 as a cation ligand and the observed inflation of KD(Mg-ATP) for S146A validates such an assignment. However, the magnitude of the Vmax effect (4000-fold) seems larger than might be expected for elimination of the liganding function. Thus, other functions for Ser-146 should be considered. In a variety of phosphotransferases, active site alcohol side chains interact with nucleotide substrate or product as hydrogen bond donors. Elimination of these side chains has an impact on catalytic efficiency that is typically larger than observed upon elimination of a cation ligand. For example, when 6-phosphofructo-2-kinase is mutated to eliminate the carboxyl group that ligands to cation of the M2+-ATP substrate, Vmax decreases by <102-fold (an effect comparable to change we reported upon mutation of mevalonate kinase's Glu-193). In contrast, mutagenesis of that enzyme to eliminate the alcohol side chain of the threonine that hydrogen bonds to ATP's beta -phosphoryl results in a >103-fold decrease in Vmax (21). Similarly, when an active site serine alcohol is eliminated from galactose-1-phosphate uridylyltransferase, Vmax drops by 7000-fold (27). This alcohol is proposed to hydrogen-bond to the nonbridging oxygen on the beta -phosphoryl of substrate UDP-glucose. In both examples, elimination of alcohols that hydrogen bond to substrate phosphoryl groups produces Vmax effects comparable to that measured for the mevalonate kinase S146A mutation. On the basis of such precedent, it seems reasonable to propose that Ser-146 functions not only as a cation ligand but also as a hydrogen bond donor to a phosphoryl oxygen of the bound substrate/product nucleotide (Fig. 6).

    ACKNOWLEDGEMENTS

We thank Matthew Riese for help in mutant plasmid construction and Drs. Kelly Chun and Jennifer Runquist for valuable advice on plasmid construction strategy. Dr. David Potter provided the initial demonstration of the utility of TNP-ATP as an active site probe for mevalonate kinase. EPR measurements were performed using the facilities of the National Biomedical ESR Center (supported by National Institutes of Health Grant RR01008).

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant DK-53766.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.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8437; Fax: 414-456-6570; E-mail: miziorko@mcw.edu.

Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M011478200

2 Residue numbering convention follows the sequence of the human enzyme.

3 Z. Fu and J. J. Kim, unpublished work.

    ABBREVIATIONS

The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; TNP-ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; ATPgamma SAP, adenosine 5'-O-[S-(acetamidoproxyl)-3-thiotriphosphate]; M2+, divalent cation; GHMP kinase, galactokinase/homoserine kinase/mevalonate kinase/phosphomevalonate kinase; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Qureshi, N., and Porter, J. W. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J. W. , and Spurgeon, S. L., eds) , pp. 47-94, John Wiley & Sons, New York
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