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J. Biol. Chem., Vol. 275, Issue 24, 17946-17953, June 16, 2000

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3-Hydroxy-3-methylglutaryl-CoA Synthase

A ROLE FOR GLUTAMATE 95 IN GENERAL ACID/BASE CATALYSIS OF C-C BOND FORMATION^*

Kelly Y. Chun, Dmitriy A. Vinarov^§, Jaroslav Zajicek^¶, and Henry M. Miziorko

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and ^¶ Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556

Received for publication, December 3, 1999, and in revised form, March 22, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Replacement of 3-hydroxy-3-methylglutaryl-CoA synthase's glutamate 95 with alanine diminishes catalytic activity by over 5 orders of magnitude. The structural integrity of E95A enzyme is suggested by the observation that this protein contains a full complement of acyl-CoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by ¹³C NMR studies, which indicate that E95A forms an acetyl-S-enzyme reaction intermediate with the same distinctive spectroscopic characteristics measured using wild type enzyme. The initial reaction steps are not disrupted in E95A, which exhibits normal levels of Michaelis complex and acetyl-S-enzyme intermediate. Likewise, E95A is not impaired in catalysis of the terminal reaction step, as indicated by efficient catalysis of a hydrolysis partial reaction. Single turnover experiments indicate defective C-C bond formation. The mechanism-based inhibitor, 3-chloropropionyl-CoA, efficiently alkylates E95A. This is compatible with the presence of a functional general base, raising the possibility that Glu⁹⁵ functions as a general acid. Demonstration of a significant upfield shift for the methyl protons of HMG-CoA synthase's acetyl-S-enzyme reaction intermediate suggests a hydrophobic active site environment that could elevate the pK_a of Glu⁹⁵ as required to support its function as a general acid.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)¹ synthase catalyzes the committed step in ketogenic and cholesterogenic pathways. Early work (1, 2) on the purified enzyme identified reaction intermediates that indicate that catalysis of HMG-CoA production involves a three-step process (Scheme 1).

View larger version (16K): [in this window] [in a new window]   Scheme 1.

Recombinant forms of both the avian (3) and human (4) enzyme have been expressed in Escherichia coli and isolated at high levels of purity. The recombinant human enzyme has been used to demonstrate that HMG-CoA synthase is the target of a potent antisteroidogenic drug (4). Mutagenesis work on the recombinant avian enzyme (3) has confirmed that Cys¹²⁹ is required to form the acetyl-S-enzyme intermediate (Scheme 1), a hypothesis that was initially advanced on the basis of protein modification by a mechanism-based inhibitor (5, 6) as well as sequence analysis of a peptide that harbors the acetyl-S-Cys¹²⁹ adduct (7).² Additionally, kinetic characterization of mutants in which His²⁶⁴ has been replaced implicates that residue (8) in binding of the second substrate, acetoacetyl-CoA. The functions of other active site residues that participate directly in catalysis have not yet been firmly established.

The sensitivity of HMG-CoA synthase activity to treatment with a carboxyl-directed modification reagent led to a preliminary observation (9), which implicated Glu⁹⁵ as a residue that is important to reaction chemistry. This report documents the crucial role of Glu⁹⁵ in catalysis and indicates which of the three steps in the reaction is compromised upon replacement of the Glu⁹⁵ carboxyl. Finally, potential functions for Glu⁹⁵ are considered, and tests aimed at discriminating between these functions are outlined. The results of implementation of these tests are interpreted and a functional assignment proposed for Glu⁹⁵ in the chemistry of C-C bond formation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Escherichia coli BL21 (DE3) and the expression vector pET-3d were purchased from Novagen (Madison, WI). E. coli strain DH5 was obtained from Life Technologies, Inc. Deoxyoligonucleotides were purchased from Operon (Alameda, CA). Qiagen (Chatsworth, CA) plasmid kits were used to isolate plasmid DNA from bacterial cultures. Qiaex (Qiagen Inc.) reagents and protocols were used for extraction of nucleic acid fragments from agarose gels. The restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA) and Amersham Pharmacia Biotech. Pfu DNA polymerase was obtained from Stratagene (La Jolla, CA). DNA sequencing was performed using an ALF automated sequencer, and the cyclosequencing kit and protocol were provided by Amersham Pharmacia Biotech. Ampicillin and isopropyl--D-thiogalactoside were purchased from U.S. Biochemical Corp. [1-¹⁴C]acetyl-CoA was purchased from Moravek Biochemicals (Brea, CA) and 3-chloro-[1-¹⁴C]propionic acid from American Radiolabeled Chemicals (St. Louis, MO). All other reagents were purchased from Sigma, Aldrich, Amersham Pharmacia Biotech, or Bio-Rad.

Methods

Sequence Homology Analysis-- All sequences used in the lineup analysis are defined in the published data bases as HMG-CoA synthases. Only sequences encoding full-length proteins were included in the analysis. Amino acid sequences were aligned using the Pileup program in the Genetics Computer Group Wisconsin Sequence Analysis Package (Genetics Computer Group, Inc., Madison, WI).

Construction of E95A HMG-CoA Synthase: Overlap Extension PCR Mutagenesis-- Point mutations were engineered in HMG-CoA synthase encoding cDNA by using the overlap extension PCR technique (10). Invariant glutamate 95 was replaced by an alanine. The PCR-amplified mutagenic fragment and nonmutagenic fragments from the parent expression plasmid were isolated by appropriate restriction and gel purification. The ligation mixture was used to transform competent DH5 cells. Mutagenic plasmid DNA was isolated from selected transformants and analyzed by restriction mapping and DNA sequencing. The verified mutant clone was transformed into competent BL21(DE3) cells for subsequent expression and isolation in the same manner as for the wild type synthase.

Isolation of E95A HMG-CoA Synthase-- The procedure (3) developed for purification of the wild type enzyme was followed for isolation of the E95A enzyme from 2-liter bacterial cultures. Protein content of the purified enzymes was estimated by the Bradford assay (11), using bovine serum albumin as the standard. The purity of the enzymes was assessed by SDS-polyacrylamide gel electrophoresis.

Synthesis of Radiolabeled 3-Chloropropionyl-CoA-- Synthesis was performed according to Miziorko and Behnke (5). Oxalyl chloride was used to activate the 3-chloro-[1-¹⁴C]propionic acid to the acyl chloride, and this intermediate was used to thioesterify CoASH. The DEAE-cellulose-purified product was assessed for concentration and purity by UV spectra and reverse-phase high pressure liquid chromatography.

Characterization of E95A Synthase-- Due to the low catalytic activity of E95A synthase, a standard spectrophotometric assay (12) could not be employed to obtain initial velocity data. The equivalent radioisotopic assay (12) was used to achieve improved sensitivity. The reaction mixture included 100 mM Tris-HCl, pH 8.2, 100 µM EDTA, 20 µM acetoacetyl-CoA, various concentrations (200-1000 µM) of [¹⁴C]acetyl-CoA (8000-12,000 dpm/nmol), and appropriately diluted E95A enzyme. The reaction was initiated by the addition of radiolabeled acetyl-CoA to the assay mixture containing the rest of the components at 30 °C. At specified time intervals, 40-µl aliquots were removed from the incubation mixture and acidified with 6 N HCl. The mixture was heated to dryness, and acid-stable radioactivity due to [¹⁴C]HMG-CoA was measured by liquid scintillation counting. Under optimal substrate conditions, activity was proportional to protein concentration, and reaction progress was linearly dependent on incubation time. However, E95A activity was not high enough to determine K_m values for the substrates.

Measurement of R·CoA binding by EPR was performed using a Varian Century-Line 9-GHz spectrometer. Samples used for recording of conventional X-band EPR spectra contained a variable concentration of HMG-CoA synthase sites (10-300 µM) in 50 mM sodium phosphate buffer, pH 7.0, and a fixed concentration of R·CoA (25 µM). The spectra were recorded at ambient temperature with a modulation amplitude of 1 G, modulation frequency of 100 kHz, and microwave power of 5 milliwatts. Field sweep was 100 G, and the time constant was 0.5 s. R·CoA bound to HMG-CoA synthase was calculated by comparing the amplitudes of high field lines of sample spectra with the corresponding lines observed for a solution containing an equal concentration of R·CoA in a buffer. Under the instrument gain and modulation amplitude conditions used to obtain these spectra, only unbound R·CoA produces a signal. Therefore, the fraction of R·CoA free in each sample was calculated by dividing the amplitude of the spectral line measured in the protein-containing samples by the amplitude of the signal measured in the absence of protein; [R·CoA]bound = ([R·CoA]total  [R·CoA]free). A Scatchard plot, fit by linear regression, was used to determine the binding constants and binding stoichiometry of R·CoA to both wild type and E95A synthases. K_d was calculated on the basis of three separate experiments. The EPR spectra of bound R·CoA were obtained at 5-G modulation amplitude and variable gain.

The stoichiometry of acetyl-CoA binding was determined by a modification of the procedure of Vollmer et al. (7). After a 5-min incubation of the enzyme (150 µg) in 100 mM sodium phosphate, pH 7.0, at 30 °C, [1-¹⁴C]acetyl-CoA (11,000 dpm/nmol) was added to bring the 100-µl reaction mixture to final concentration to 200-1000 µM. Unbound acetyl-CoA was removed using a G-50 centrifugal column equilibrated with 20 mM sodium phosphate buffer, pH 7.0. Protein in the recovered samples was estimated by the Bradford assay, and radioactivity was determined by liquid scintillation counting.

Stoichiometry of covalent acetylation was determined by a modification of the procedures of Miziorko et al. (1). The 40-µl aliquots of incubation mixture (100 mM potassium phosphate, pH 7.0, containing saturating levels of [1-¹⁴C]acetyl-CoA (8000-12,000 dpm/nmol) and 40 µg of enzyme (1 mg/ml final concentration)) were treated with 1 ml of ice-cold 10% trichloroacetic acid. The denatured protein was transferred to a glass fiber filter. The filters were washed extensively with ice-cold 10% trichloroacetic acid and 50 mM sodium pyrophosphate in 500 mM HCl and once with cold absolute ethanol. Filters were dried, and radioactivity was determined by liquid scintillation counting.

Acetyl-CoA hydrolase activity of wild type and mutant synthases was measured as reported previously (1) by monitoring enzyme-dependent depletion of [1-¹⁴C]acetyl-CoA after conversion of residual substrate to acid-stable [¹⁴C]citrate, using excess citrate synthase and oxaloacetate.

Single turnover reaction was measured by combining several techniques described above. Enzyme (3 nmol) was incubated with a saturating concentration of [1-¹⁴C]acetyl-CoA (500-1000 µM, 12,000 dpm/nmol). The 100-µl incubation mixture was spun through a 2-ml G-50 centrifugal column equilibrated with 20 mM sodium phosphate to remove the unbound acetyl-CoA. Approximately a 10-fold excess (over enzyme sites) of unlabeled second substrate (acetoacetyl-CoA) was added to the acetyl-enzyme intermediate recovered in the filtrate to drive the reaction to completion. Over time, 30-µl aliquots of this reaction mix were removed from the incubation mixture and acidified with 6 N HCl. The mixture was heated to dryness, and acid-stable radioactivity due to condensation product, [¹⁴C]HMG-CoA was measured by liquid scintillation counting. At each time point, additional 30-µl aliquots were treated with 1 ml of ice-cold 10% trichloroacetic acid. The denatured protein was transferred to a glass fiber filter. The filters were washed extensively with ice-cold 10% trichloroacetic acid and 50 mM sodium pyrophosphate in 500 mM HCl and once with cold absolute ethanol. Filters were dried, and radioactivity was determined by liquid scintillation counting.

Formation of Covalent Adducts between HMG-CoA Synthase and the 3-Chloro-[1-¹⁴C]propionyl-CoA-- 20-µl aliquots (20 µg of enzyme) of an incubation mixture containing (3-chloro-[1-¹⁴C]propionyl-CoA (12 nmol, 16,800 dpm/nmol) and enzyme (1.4 nmol) in 100 mM Tris-HCl, pH 8.2, 100 µM EDTA) were removed and treated with 1 ml of ice-cold 10% trichloroacetic acid at specified time intervals. The denatured protein was transferred to a glass fiber filter. The filters were washed extensively with ice-cold 10% trichloroacetic acid and 50 mM sodium pyrophosphate in 500 mM HCl and once with cold absolute ethanol. Filters were dried, and radioactivity was determined by liquid scintillation counting.

NMR Methodology-- ¹H, ¹³C () half-filtered experiments were performed using a Varian Unity Plus 600 spectrometer operating at 599.885 MHz for ¹H. All spectra were recorded at 21 °C and referenced to DSS ( = 0 ppm). Residual HOD resonance was suppressed by lower-power presaturation. ¹³C decoupling in ₂ was carried out using the GARP decoupling scheme. The spectra were obtained using a ¹H spectral width of 6800 Hz; 16,000 data points were collected.

¹³C NMR (proton-decoupled) experiments were performed using a Bruker AC-300 instrument operating at 75.469 MHz for ¹³C. All spectra were recorded at 21 °C and referenced to tetramethylsilane. A sweep width of 16,000 Hz was used, and 16,000 data points were collected. Signal acquisition employed a 35-degree pulse angle and a 2-s delay between transients. A typical spectrum of ¹³C-enriched acetyl-CoA, measured in samples with a 2:1 substrate/enzyme site ratio, required 1.5-5 h of data collection (1500-5000 transients). For spectra shown in the figures, the collected data were zero-filled to 64,000 points and then processed with 5-Hz line broadening to improve signal/noise ratio. ¹³C-enriched acetyl-CoA was lyophilized and dissolved in 100% D₂O prior to running the spectra. HMG-CoA synthase samples were buffer-exchanged into 10 mM sodium phosphate, pH 7.0, using Centricon-25 membrane cones. After concentration to an appropriate site concentration (2.25 mM for the ¹H and 1 mM for the ¹³C experiments), the samples were lyophilized and redissolved (without significant loss of activity) in an appropriate volume of either deionized water supplemented with 20% D₂O for internal lock (¹³C experiment) or 100% D₂O (¹H experiment) prior to mixing with acetyl-CoA and running the spectra.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mutagenesis Strategy, Expression, and Isolation of HMG-CoA Synthase E95A-- HMG-CoA synthase exhibits time- and concentration-dependent inactivation by the carboxyl-directed reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Substantial protection against activity loss is observed when the experiment is performed in the presence of substrate acetoacetyl-CoA. These observations, together with the precedent for participation of carboxyl groups in mechanistically analogous C-C bond-forming reactions catalyzed by citrate synthase (13) and fructose 1,6-bisphosphate aldolase (14), prompted evaluation of the functional roles of invariant acidic residues in HMG-CoA synthase. Alignment of deduced amino acid sequences for HMG-CoA synthase (Fig. 1; 15 eukaryotic and two prokaryotic proteins are included) indicates that Glu⁹⁵ is invariant. Replacement of this residue by alanine has been accomplished by PCR mutagenesis.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Alignment of deduced amino acid sequences for HMG-CoA synthases to indicate the location of the mutated glutamic acid. All full-length HMG-CoA synthase sequences were obtained from public data bases. Alignment was generated by using the Pileup program of Genetics Computer Group Sequence Analysis Software. The shaded column indicates that Glu95 is invariant among these species. c and m specify cytosolic and mitochondrial isoforms, respectively. Accession numbers are as follows: Methanobacterium thermoautotrophicum, AE000857; Borrelia burgdorferi, BB0683; Schizosaccharomyces pombe, U32187; Saccharomyces cerevisiae, P54871; Arabidopsis thaliana, X83882; Pinus sylvestris, X96386; Mus musculus (m), P54869; Rattus norvegicus (m), P22791; Sus scrofa (m), U90884; Homo sapiens (m), P54868; Blattella germanica (c1), P54961; Blattella germanica (c2), P54870; Gallus gallus (c), P23228.

Expression constructs that encode E95A synthase support production of the mutant protein in a soluble form at levels comparable with that observed for wild type enzyme. E95A synthase is isolated using the same protocol as reported for wild type enzyme (3); the mutant enzyme displays identical chromatographic properties and exhibits a comparable level of homogeneity (Fig. 2) and stability.

View larger version (109K): [in this window] [in a new window]   Fig. 2.   E95A synthase at various stages of purification. Lane 1, molecular mass markers: phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Lane 2, total cell lysate of E. coli containing the plasmid encoding E95A synthase. Lane 3, supernatant after centrifugation of bacterial extract at 46,000 × g. Lane 4, 40% (NH4)2SO4 fraction. Lane 5, Fast-Q eluate. Lane 6, purified wild type HMG-CoA synthase.

Initial Characterization and Tests of Structural Integrity-- Using a sensitive radioactive activity assay, it is possible to estimate that E95A is diminished in catalytic activity by 5 orders of magnitude (Table I). This low residual activity precluded measurements under suboptimal conditions. Thus, neither estimates of K_m for acetyl-CoA or acetoacetyl-CoA nor pH/rate profiles can be determined. The lack of these parameters eliminates any ability of making the comparisons most commonly used in estimating whether a mutant retains some wild type enzyme traits. This dilemma underscores a basic question that must be addressed before attempting to deduce the mechanistic basis for any large decrease in catalytic efficiency upon mutation of a single amino acid. May the large effect be simply attributed to a structural alteration that characterizes the mutant protein? If no major perturbation in tertiary structure accounts for the observed decrease, then the 5-order of magnitude diminution in activity would certainly implicate Glu⁹⁵ as an important participant in reaction chemistry. To address this issue, different spectroscopic tools that have been previously developed and productively employed (3, 15) to evaluate the C129S mutant (which lacks the ability to form the acetyl-S-enzyme reaction intermediate) have been utilized in characterizing the structure of E95A.

The spin-labeled substrate analog, R·CoA, binds to the active site as a competitive inhibitor with respect to acetyl-CoA and produces an ESR signal indicating strong immobilization of the probe on a dimeric protein of 116 kDa. Additionally, binding of the probe to enzyme eliminates the signal due to free spin-label and facilitates quantitation of binding. Scatchard analysis (Fig. 3) allows these data to be straightforwardly analyzed to produce binding stoichiometry and K_d estimates for R·CoA. These parameters, listed in Table I, compare favorably with values previously reported for wild type enzyme. Binding stoichiometry (calculated on the basis of a 57.6-kDa subunit) indicates that E95A contains a full complement of functional acyl-CoA binding sites. K_d for the spin-labeled probe is 4-fold tighter than measured for wild type enzyme, further arguing that active site structure is not seriously altered. Finally, by analysis of the spectral features of the bound spin probe, the rotational dynamics of the heterocyclic acyl group can be evaluated (16). This group remains substantially immobilized when R·CoA binds to E95A. The estimate of correlation time (_c = 35 ns; Table I) is indistinguishable from the value reported for wild type enzyme (3, 17). These observations indicate that E95A synthase exhibits no significant alteration of secondary or tertiary structure that would increase local mobility within the active site, detectable as more rapid tumbling of the heterocyclic reporter group. It seems quite probable that, if the active site binding of the acyl-CoA substrate analog remains unchanged, the overall tertiary structure is also intact.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Scatchard plot of R·CoA binding to E95A HMG-CoA synthase. Data were obtained by mixing various amounts of E95A synthase (10 to 300 µM) with a fixed concentration of R·CoA (25 µM). The amount of free ([R·CoA]f) and bound ([R·CoA]b) spin label were determined by measuring the amplitude of the high field ESR spectral line as indicated under "Experimental Procedures." The data were normalized with respect to enzyme concentration ([E]).

In addition to testing for the characteristic binding of an acyl-CoA, the ability of E95A to form a covalent reaction intermediate that also possesses distinctive spectroscopic traits can be evaluated. Vinarov et al. (15) have successfully employed ¹³C NMR spectroscopy to show that transfer of an acetyl moiety from CoA to form the acetyl-S-enzyme reaction intermediate results in marked changes in the magnetic environment of both acetyl carbons, measured as upfield shifts of 20 and 7 ppm for C-1 (thioester carbonyl) and C-2 (methyl) carbons, respectively. C129S synthase, which has an unperturbed acyl-CoA binding site but fails to transfer the acetyl group from CoA to enzyme, will not produce the upfield shifts that characterize wild type enzyme. In contrast, when E95A is incubated with [1,2-¹³C]acetyl-CoA to form [1,2-¹³C]acetyl-S-enzyme, the sample produces a ¹³C NMR spectrum (Fig. 4) identical to wild type synthase. The magnitude of the upfield shifts of the ¹³C doublet resonances attributable to C-1 and C-2 of the covalently bound acetyl group (Table I) is comparable with that measured in wild type enzyme control experiments. These observations rigorously demonstrate that there is a very high degree of similarity between the active site environments of the acetyl-S-enzyme reaction intermediates formed from wild type and E95A synthases.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   [1,2-13C]acetyl-CoA binding to wild type and E95A HMG-CoA synthases. 13C NMR spectra shown represent the down-field (210-180 ppm) and upfield (38-22 ppm) regions of 2.0 mM [1,2-13C] acetyl-CoA in 10 mM KPi, pH 7.0 (A); 2.0 mM [1,2-13C]acetyl-CoA plus 1 mM (sites) wild type HMG-CoA synthase (B); 2.0 mM [1,2-13C] acetyl-CoA plus 1 mM (sites) E95A HMG-CoA synthase (C). The wild type and mutant synthases were buffer-exchanged into 10 mM KPi buffer, pH 7.0, lyophilized, and dissolved in deionized water supplemented with 20% D2O for internal lock. Acetyl-CoA was added to the enzyme samples just prior to data collection. Spectra were obtained on a Bruker-300 MHz NMR spectrometer. All spectra were recorded at 21 °C. For spectral display, the free induction decays were processed with 5-Hz line broadening. Chemical shifts are referenced to tetramethylsilane. Spectra shown were obtained with 1500, 5000, and 5000 scans for A, B, and C, respectively.

Comparison of Catalysis of Partial Reactions by Wild Type and E95A HMG-CoA Synthases-- As indicated in Scheme 1, HMG-CoA biosynthesis is initiated by formation of a Michaelis complex and covalent acetyl-S-enzyme intermediate. Next, a C-C bond forms upon condensation with the second substrate. Finally, a hydrolytic reaction releases product. The efficiency of the initial partial reaction can be evaluated by measurement of binary acetyl-CoA*enzyme and covalent acetyl-S-enzyme species. Likewise, efficiency of the terminal product release is monitored by the measuring the rate of the analogous hydrolysis reaction, which occurs when enzyme is incubated with acetyl-CoA in the absence of second substrate.

Centrifugal gel filtration (7) affords an estimate of enzyme's occupancy by either acetyl-CoA*enzyme Michaelis complex or covalent acetyl-S-enzyme reaction intermediate. When binding stoichiometries measured for wild type and E95A synthases are compared (Table II), it is clear that the enzymes are equivalent in this respect. Moreover, the component of the binding stoichiometry due to covalent reaction intermediate can be assigned on the basis of trichloroacetic acid precipitation experiments. Results of such measurements (Table II) also indicate that these enzymes are indistinguishable in efficiency of production of reaction intermediate, accounting for the ability to straightforwardly measure the ¹³C NMR spectrum of E95A's reaction intermediate (Fig. 4). The observation of identical chemical shifts for the ¹³C signals attributable to the acetyl group of the wild type and E95A reaction intermediates represents a strong biophysical argument that these species are functionally equivalent. The chemical similarity of these species can also be evaluated. Table III shows that the radiolabeled acetyl-S-enzyme reaction intermediate formed using wild type or E95A synthase can be trapped in comparable yield. For both species, the radiolabeled adduct can be completely labilized by incubation with neutralized hydroxylamine (100 mM) or by exposure to performic acid vapor (Table III). A negative control is afforded by formic acid vapor treatment, which will not efficiently hydrolyze thioester adducts; the radiolabeled adducts are minimally affected by exposure to formic acid using conditions sufficient for complete labilization by performic acid. These equivalent properties of wild type and E95A acetyl-S-enzyme reaction intermediates, as measured using both biophysical and chemical approaches, clearly indicate that the decrease in E95A's catalytic efficiency is not due to any impairment in the early phase of the reaction.

In the absence of second substrate, E95A synthase catalyzes acetyl-CoA hydrolysis. HMG-CoA synthases do not hydrolyze acetyl-CoA by catalyzing a simple nonspecific addition of water across the thioester linkage between acetyl and CoA moieties in the Michaelis complex that forms and persists in the absence of the second substrate. Instead, as demonstrated by our previous studies (3), the acetyl-CoA hydrolysis partial reaction requires prior formation of a specific adduct between the acetyl C-1 carboxyl and the thiol of Cys¹²⁹ in the active site. The enzyme catalyzes no substantial acetyl-CoA hydrolysis unless the acetyl-S-Cys¹²⁹ adduct is first formed. This adduct contains the same C-S bond that is ultimately cleaved in the hydrolytic release of product after the condensation event. While hydrolysis efficiencies of acetyl-S-enzyme and CoAS-HMG-S-enzyme may differ, since the acetyl-CoA hydrolysis partial reaction directly measures cleavage of the same bond involved in terminal product release, such activity clearly reflects this late step in the overall reaction. Both in terms of catalytic efficiency of hydrolysis (V_m = 0.010 units/mg) and Michaelis constant (K_m acetyl-CoA = 14 µM), E95A is equivalent to wild type enzyme (Table II). Thus, these data, as well as results of single turnover experiments presented below, indicate that impairment of the terminal hydrolysis reaction does not account for E95A's catalytic deficiency. Having eliminated the likelihood that initial or terminal phases of the overall reaction are compromised, it becomes necessary to evaluate the condensation step in the reaction.

One approach to evaluating differences between wild type and E95A synthases involves measurement of the efficiency of single condensation reaction turnovers. This is accomplished by preincubation of enzyme with [¹⁴C]acetyl-CoA and isolation, via centrifugal gel filtration, of enzyme stoichiometrically populated with a [¹⁴C]acetyl moiety. This sample is mixed with an excess of the second substrate, acetoacetyl-CoA. After specified incubation times, aliquots are either subjected to trichloroacetic acid precipitation (detecting radiolabeled acyl-S-enzyme intermediates) or brought to 6 N HCl and taken to dryness (detecting radiolabeled condensation intermediate or product). When the reaction is performed with wild type enzyme, there is, as expected, efficient rapid turnover (Fig. 5) to quantitatively convert the [¹⁴C]acetyl moiety (stoichiometry of 0.6/site) into acid-stable ¹⁴C-labeled condensation product, HMG-CoA (stoichiometry of 0.6/site). When E95A is used to perform the single turnover experiment, the results contrast sharply. No substantial amounts of acid-stable [¹⁴C]HMG-CoA are formed, indicating a defect in C-C bond formation. Accordingly, the initial stoichiometry of trichloroacetic acid-precipitable acetyl-S-enzyme reaction intermediate (0.7/site) does not change upon various time periods of incubation with second substrate (Fig. 5). The results clearly indicate that E95A's catalytic impairment is due to a deficiency in the chemistry of the condensation event. If there had been a defect in product hydrolysis, covalently bound enzyme-S-[¹⁴C]HMG-SCoA would accumulate, and acid-stable, trichloroacetic acid-precipitable ¹⁴C radioactivity would be readily detectable. No such intermediate accumulates. Since these observations clearly indicate that E95A is defective in C-C bond formation, it remains to be determined whether a precise chemical step in condensation can be implicated as accounting for this defect.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Single turnover reactions of wild type and E95A synthase. Each wild type and E95A enzyme sample (25 µM) was incubated with a saturating concentration of [1-14C]acetyl-CoA (500 µM, 12,000 dpm/nmol). Unbound acetyl-CoA was removed using a size exclusion centrifugal column. Excess unlabeled second substrate was added to the filtrate containing acetyl-S-enzyme intermediate to drive the reaction to completion. The reaction mixture was monitored over time for formation of condensation product, HMG-CoA, and for the remaining level of covalent substrate/product-enzyme intermediate. , acid-stable (6 N HCl) 14C radioactivity produced upon formation of condensed product, HMG-CoA; , trichloroacetic acid-precipitable counts due to covalent enzyme-bound intermediates.

Alkylation of Wild Type and Mutant HMG-CoA Synthases by the Mechanism-based Inhibitor, 3-Chloropropionyl-CoA-- Previous work from our laboratory (5, 6) has demonstrated that cysteine 129 is selectively alkylated by the substrate analog, 3-chloropropionyl-CoA. Additional studies (18) suggest that the process reflects a mechanism-based process, with a general base on the enzyme catalyzing deprotonation of C-2 of the analog's acyl group, a reaction comparable with the deprotonation step that precedes productive condensation between the enzyme's normal substrates. After C-2 of the inhibitor is deprotonated, elimination of chloride and production of acryloyl-SCoA ensues. This species contains a double bond conjugated to the thioester functionality, accounting for its high reactivity in alkylating the nucleophilic sulfur atom of Cys¹²⁹ (Scheme 2).

View larger version (5K): [in this window] [in a new window]   Scheme 2.

A survey of the ability of [1-¹⁴C]3-chloropropionyl-CoA to alkylate various HMG-CoA synthases has been performed. The results (Table IV) indicate that no alkylation of serum albumin (nonspecific negative control sample) or C129S (lacking the alkylation site) is observed upon the brief incubation with inhibitor that is sufficient to eliminate catalytic activity. Only upon long incubations (60 min) of C129S with inhibitor does minor alkylation of a secondary target (19) become detectable. In contrast, both wild type and E95A enzymes are rapidly alkylated by the reagent (Table IV), indicating the enzymatic apparatus (general base) responsible for converting reagent to a reactive species is intact in both proteins. If the results are interpreted to suggest that E95A retains a functional general base, the issue of a functional assignment for the carboxyl group of Glu⁹⁵ persists.

NMR Characterization of the Active Site Environment of HMG-CoA Synthase-- Before attempting to deduce a role for Glu⁹⁵ on the basis of the array of data presented above, it seems prudent to consider factors that may influence the reactivity of the Glu⁹⁵ carboxyl group. Active site functional groups involved in C-C bond formation must be closely juxtaposed to the acetyl moiety of the acetyl-S-enzyme reaction intermediate. If the C-2 protons of this intermediate can be detected, then comparison of their environment with that of the corresponding protons in an aqueous solution acetyl-CoA can be accomplished and could be informative. HMG-CoA synthase is a dimer of 58-kDa subunits, raising some concern over whether an NMR approach would be productive in detection of the acetyl protons. Despite this concern, our ability to detect [¹³C]acetyl-S-enzyme (15) encouraged an attempt to utilize NMR to address this issue.

The C-2 protons have been successfully detected on [1,2-¹³C]acetyl-S-enzyme by use of a ¹H,¹³C-edited NMR approach. At the top of Fig. 6, the ¹³C-edited spectrum of [1,2-¹³C]acetyl-CoA is depicted; the upfield (2.32 ppm) resonance is attributable to the methyl protons. An unedited ¹H spectrum of [1,2-¹³C]acetyl-S-enzyme is shown as the middle trace of Fig. 6, indicating the complexity of detecting the acetyl group in the presence of the array of protonated amino acids in a 522-amino acid protein. The bottom trace in Fig. 6 corresponds to a ¹³C-edited spectrum of the same sample used to produce the middle trace. The resonance attributable to the methyl protons of [1,2-¹³C]acetyl-S-enzyme is readily detectable and is shifted upfield by about 0.5 ppm from the position of the comparable signal measured using aqueous acetyl-CoA (top trace). An upfield shift of this type upon localization of a metabolite within an enzyme's active site is commonly attributed (20) to the change of environment of the protons from a high dielectric constant aqueous medium to a hydrophobic, low dielectric active site.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   600-MHz 1H,13C (2) half-filtered spectra of [1,2-13C]acetyl-CoA binding to wild type HMG-CoA synthase. 1H spectra shown in A and C were acquired with a 13C (2) half-filter. Samples contained 2.0 mM [1,2-13C]acetyl-CoA in 10 mM KPi, pH 7.0 (A); 2.0 mM [1,2-13C]acetyl-CoA plus 2.25 mM (sites) wild type HMG-CoA synthase (B); 2.0 mM [1,2-13C]acetyl-CoA plus 2.25 mM (sites) wild type HMG-CoA synthase (C). Resonances corresponding to the methyl protons on the C-2 of the acetyl-CoA in buffer (A) and the C-2 of the acetyl-S-enzyme reaction intermediate (C) are indicated with an arrow.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

There is clearly a dramatic difference between the relative efficiencies with which wild type HMG-CoA synthase and the E95A mutant catalyze HMG-CoA production. In contrast, these proteins exhibit a large number of similarities when only the early or late steps along the reaction coordinate are compared. The similarities in capacity for acetyl-CoA binding or acetyl-S-enzyme formation, whether evaluated by chemical or biophysical approaches, leave no doubt that steps leading to the condensation event are unimpaired. Likewise, comparable efficiencies in the late hydrolysis and product release steps in the reaction, whether monitored by release of soluble product in single turnover experiments or by following the acetyl-CoA hydrolysis partial reaction, indicate a defect in the upstream condensation step.

An array of data support the assignment to Glu⁹⁵ of a role in C-C bond formation. The degree to which the available data specifically support a direct role for Glu⁹⁵ in general acid/base catalysis remains to be addressed. Such assignments are frequently made on the basis of diminutions in catalytic efficiency that are substantially more modest in magnitude than we observe upon elimination of HMG-CoA synthase's Glu⁹⁵. These practices underscore the importance of correctly evaluating the intrinsic contribution of a general acid/base to catalysis. One straightforward estimate, generated before the widespread application of site-directed mutagenesis to enzymology, was documented by Meloche's laboratory (21) in work on ketodeoxygluconate 6-phosphate aldolase. This enzyme exhibits a >10⁵-fold difference in V/K values for cleavage of ketodeoxygluconate 6-phosphate versus ketodeoxygalactonate 6-phosphate. The difference is attributed to efficiency of general base catalysis versus an uncatalyzed reaction; it represents a realistic benchmark that should be useful to enzymologists as they offer interpretations to explain the magnitude of observed mutagenic effects. Using this benchmark, HMG-CoA synthase's Glu⁹⁵ does indeed qualify for consideration as a direct participant in general acid/base catalysis.

In attempting to distinguish between whether Glu⁹⁵ functions as a general acid or general base, it seems useful to draw on some precedents generated with citrate synthase, which catalyzes an analogous condensation reaction. The combined approaches of mutagenesis (13, 22) and x-ray crystallography (23) have led to the assignment of porcine citrate synthase's Asp³⁷⁵ as the general base involved in acetyl-CoA deprotonation and His³²⁰ as the acid that protonates oxaloacetate's C-2 ketone. Elimination of either side chain produces a substantial diminution in condensation activity. Thus, assignment of a precise function to HMG-CoA synthase's Glu⁹⁵ requires more than just kinetic characterization of mutants. If the mechanism (18) outlined for enzyme alkylation by chloropropionyl-CoA (i.e. transient production of a reactive acryloyl-SCoA intermediate) is correct, the sensitivity of E95A to this reagent seems significant. It is difficult to reconcile such efficient alkylation unless an efficient general base catalyst is present to support production of a reactive species from the intrinsically unreactive 3-chloropropionyl-CoA. While it remains possible that a surrogate side chain fulfills the general base function in E95A, the simplest interpretation of these data would discount the role of Glu⁹⁵ as a general base and require a different assignment.

The remaining chemical role for which this residue may qualify is that of general acid. Such speculation raises the issue of Glu⁹⁵'s pK_a value, which might be expected to be too low to support efficient protonation of the C-3 keto group of acetoacetyl-CoA. Nevertheless, roles for carboxylic acid side chains as general acids have been documented in a variety of enzymic reactions (24-26). Factors such as hydrogen bonding networks (24), hydrophobic active site environments (25), and pK_a changes upon formation of covalent reaction intermediates (26) have been proposed to account for the elevation of carboxyl pK_a from the values of 4-5 typically measured in aqueous solution. The latter two factors may be pertinent in the context of HMG-CoA synthase. There certainly is ample proof that the acetyl-S-enzyme covalent reaction intermediate is situated in a hydrophobic environment. The upfield shifts measured both for methyl protons of acetyl-S-enzyme (Fig. 6) and for the C-1 thioester carbonyl of this reaction intermediate (15) suggest a low dielectric active site environment. This hypothesis has been confirmed in model studies that demonstrate upfield ¹³C NMR shifts of the acetyl groups of N,S-diacetylcysteamine upon change from a high to low dielectric constant solvent (15). On the basis of these observations, an increase of several pK units does not seem unreasonable for the Glu⁹⁵ carboxyl group; a perturbation of this magnitude would be compatible with a protonated carboxyl side chain. While such speculation invites additional experimental tests, the provisional assignment of Glu⁹⁵ as a general acid is compatible with all available data and merits consideration.

	ACKNOWLEDGEMENTS

We acknowledge the assistance of Dr. Chakravarthy Narasimhan in ESR measurements and ESR spectral analysis. The spin-label 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-21491.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.

Predoctoral fellow of the American Heart Association.

^§ Postdoctoral fellow of the American Heart Association.

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.

Published, JBC Papers in Press, March 30, 2000, DOI 10.1074/jbc.M909725199

² The residue numbering convention follows the sequence of the avian and human cytosolic proteins.

	ABBREVIATIONS

The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; R·CoA, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl-CoA; PCR, polymerase chain reaction; ESR, electron spin resonance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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