©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evaluation of Cysteine 266 of Human 3-Hydroxy-3-methylglutaryl-CoA Lyase as a Catalytic Residue (*)

(Received for publication, March 31, 1995; and in revised form, May 18, 1995)

Jacqueline R. Roberts , Chakravarthy Narasimhan , Henry M. Miziorko (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The role of cysteine 266 in human 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, a residue that is homologous to a cysteine mapped to the active site of prokaryotic HMG-CoA lyase by protein chemistry approaches, has been investigated by site-directed mutagenesis. Both the wild-type human enzyme and a C323S variant, in which a regulatory sulfhydryl has been eliminated without any negative effect on catalytic activity (Roberts, J. R., Narasimhan, C., Hruz, P. W., Mitchell, G. A., and Miziorko, H. M.(1994) J. Biol. Chem. 269, 17841-17846), were used as models. Mutant enzymes C266A, C266A/C323S, C266S, and C266S/C323S were overexpressed in Escherichia coli and purified to homogeneity. In all cases, kinetic characterization indicated that the K value for HMG-CoA was not substantially different from the value measured using wild-type human lyase, suggesting that no serious structural perturbation occurs upon replacing Cys-266. A dissociable divalent cation (Mn or Mg), which is required for activity in both native and C323S enzymes, is also an essential component for activity in each of the Cys-266 mutants. The structural integrity of the human mutants was further indicated by Mn binding studies, which demonstrate similarities not only in the activator cation binding stoichiometries, but also in the K values for Mn as determined for wild-type and mutant C266A or C266S proteins. Purified C266A and C266A/C323S mutants both displayed 1.3 10-fold diminution in specific activity, while the k value was diminished in both C266S and C266S/C323S by 9.9 10-fold. This large diminution in catalytic efficiency in enzyme variants that display no substantial structural perturbations is in accord with an active-site assignment to Cys-266 and qualifies its sulfhydryl group for consideration as a component of the catalytic apparatus.


INTRODUCTION

3-Hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) catalyzes the cleavage of HMG-CoA()to form acetyl-CoA and acetoacetate. This reaction accounts for the formation of ketone bodies, an alternate source of fuel during times of fasting or starvation(1) . In addition, HMG-CoA lyase functions in the final steps of leucine catabolism(2) . A deficiency in HMG-CoA lyase can result in the inherited metabolic disease hydroxymethylglutaric aciduria(3) , which, in some populations, can represent 16% of hereditary organic acidurias(4) . Elucidation of the cDNA sequences that encode a number of eukaryotic enzymes, including the human, chicken, and mouse proteins, has been reported(5, 6) . The sequence of HMG-CoA lyase from Pseudomonas mevalonii has also been deduced(7) . Comparison of the deduced peptide sequences reveals that the human lyase polypeptide exhibits 87% identity to mouse, 82% to chicken, and 52% to P. mevalonii lyases.

Two reactive cysteines have been identified on the basis of mechanistic and protein chemistry studies on HMG-CoA lyases. Work with the avian enzyme indirectly implicated a cysteine in in vitro regulation of the activity of eukaryotic HMG-CoA lyase by controlling the thiol/disulfide state of the enzyme(8) . A hyperreactive cysteine was mapped to the C-terminal region of the avian protein and was found to be capable of forming an intersubunit cross-link with the corresponding cysteine on the other subunit of this dimeric enzyme(8) . This cysteine is conserved in both human and mouse HMG-CoA lyases, but not in the bacterial enzyme. Interestingly, the activity of the bacterial enzyme is not highly dependent on thiol-reducing reagents, as are the eukaryotic counterparts(9) . Mutation of this reactive cysteine (C323S)()in the human enzyme did not impair catalytic function, but led to a complete loss of the enzyme's ability to form an intersubunit cross-link and to substantial diminution in the requirement for thiol reagents to maintain activity(10) .

A second reactive cysteine has been mapped to the active site. Utilizing both bacterial and avian lyases, Hruz et al.(11) established that the substrate analog 2-butynoyl-CoA stoichiometrically modifies the enzyme. Protein chemistry studies on the modified prokaryotic protein established Cys-237 as the target. Alignment of the available amino acid sequences for HMG-CoA lyases suggests that the labeled cysteine corresponds to Cys-266 of the human enzyme.

Recently, a recombinant system for expression and isolation of human HMG-CoA lyase was developed(10) . Utilizing this system, site-directed mutagenesis was employed to replace, in both wild-type and C323S enzymes, the Cys-266 sulfhydryl group. This report describes our strategy for evaluating the structural integrity of the isolated mutant proteins, which is a prerequisite to any meaningful interpretation of the results of the accompanying kinetic characterization. The results represent a significant confirmation of the active-site assignment prompted by the original affinity labeling studies and implicate human lyase Cys-266 in the chemistry of HMG-CoA cleavage.


EXPERIMENTAL PROCEDURES

Materials

[C]Acetic anhydride was purchased from DuPont NEN. Q-Sepharose anion-exchange resin and Superose-12 (preparatory grade) resin were products of Pharmacia Biotech Inc. Phenyl-agarose was purchased from Sigma. Restriction enzymes were a product of New England Biolabs Inc. Qiaex and Qiagen were purchased from QIAGEN Inc. (Chatsworth, CA). HMG-CoA was synthesized from the anhydride, prepared from the free acid (Fluka) according to Goldfarb and Pitot (12) . All other chemicals were reagent-grade.

Methods

Enzymatic Preparation of 3-[C]Hydroxy-3-methylglutaryl-CoA

Radiolabeled HMG-CoA was enzymatically synthesized (13) from acetoacetyl-CoA and radiolabeled [1-C]acetyl-CoA(14) . The product (6000 dpm/nmol) was purified by DEAE-Sephadex chromatography and isolated in a salt-free form using a Sep-Pak reverse-phase cartridge(8) .

Construction of Mutants Containing Substitutions for Cysteine 266

Construction of the C266S, C266S/C323S, C266A, and C266A/C323S mutations was performed as follows. Either the pTrcHL-1 or pTrcHL-C323S plasmid (10) was digested with NcoI and BamHI, and the resulting 925-base pair fragment containing the coding region corresponding to the mature matrix form of the protein was isolated and purified using the Qiaex kit and protocol (QIAGEN Inc.). pTrc99A was digested with NcoI and BamHI; the resulting 4.1-kilobase vector fragment was purified as described above. The 925-base pair fragments, containing either the wild-type coding sequence or C323S, were digested with PstI and AccI, which have unique restriction sites within the coding sequence. The double digestion generated three fragments with sizes of 680, 95, and 150 base pairs. The C266S or C266S/C323S and the C266A or C266A/C323S mutations were generated with a synthetic oligonucleotide duplex, spanning the 95-base pair region between the unique PstI and AccI restriction sites. The synthetic cassette was generated by annealing six oligonucleotides (three for each strand). The cysteine codon was altered to either serine (TCT) or alanine (GCT). The generation of a viable plasmid encoding the desired mutation at position 266 of the human HMG-CoA lyase gene was then accomplished by a four-way ligation of the following: the appropriate synthetic cassette, the isolated 680-base NcoI/PstI fragment, the isolated 150-base AccI/BamHI fragment, and the 4.1-kilobase fragment isolated from the NcoI/BamHI-digested pTrc99A plasmid. The resulting expression vectors, pTrcHL-C266S, pTrcHL-C266S/C323S, pTrcHL-C266A, and pTrcHL-C266A/C323S, were sequenced in both directions (15) to confirm that only the appropriate mutations were encoded in the region where the synthetic cassette replaces the wild-type sequence.

Purification of Human Lyase Mutants

Recombinant wild-type human lyase, C323S lyase, and the C266A, C266S, C266A/C323S, and C266S/C323S variants were expressed and purified as described by Roberts et al.(10) . All mutants purified identically to the wild-type enzyme. The isolated enzymes were >95% pure. Protein concentrations were determined by the Bradford method (16) using bovine serum albumin as a standard. SDS-polyacrylamide gel electrophoresis was run under denaturing conditions as described by Laemmli (17) utilizing an 11% acrylamide running gel and a 4.5% acrylamide stacking gel.

Kinetic Characterization of Human HMG-CoA Lyase Mutants

Enzymatic activity of HMG-CoA lyase and the engineered variants was determined using the citrate synthase-coupled assay of Stegink and Coon (18) as modified by Kramer and Miziorko (19) except that 100 µM HMG-CoA was used to initiate the reaction. When the K value of Mn was determined, all components of the assay mixture, including buffers, were treated with Chelex 100 to remove trace metals.

The pH/rate profiles for wild-type and C323S lyases were measured as follows. Enzyme was incubated for 15 min at room temperature in the presence of 20 mM dithiothreitol and then placed on ice until needed. After addition of the reduced lyase to the assay mixture, the reaction was immediately initiated with HMG-CoA, thus minimizing any instability related to changes in pH. The buffer employed in the assay mixture was either 100 mM Tris-HCl, 0.10 mM EDTA for pH values between 6.6 and 8.9 or 100 mM glycine, 0.10 mM EDTA for pH values between 8.7 and 10.0. The V value for HMG-CoA lyase was determined at the specified pH values by linear regression analysis of the Lineweaver-Burk plots. The pH/rate profile data were fit using a nonlinear regression analysis algorithm(20) .

The pH dependence of C266A/C323S lyase was measured using a radioactive assay (21) in order to improve sensitivity. The reaction mixture contained the appropriate buffer (specified above), 10 mM MgCl, HMG-CoA lyase (8.2 µg of C266A/C323S) preincubated with 20 mM dithiothreitol, and 100 µM [C]HMG-CoA in a final volume of 200 µl. The reaction was initiated by addition of the enzymatically synthesized [C]HMG-CoA (6000 dpm/nmol) at 30 °C. At various time points, aliquots were removed and acidified with 6 N HCl. After heating to dryness, the acid-stable radioactivity attributable to [C]HMG from unreacted substrate was determined by liquid scintillation counting. Depletion of acid-stable radioactivity is a measure of enzymatic cleavage of substrate to form acid-volatile product (acetoacetate). The radioactive assay, under standard pH conditions (Tris-HCl, pH 8.2), was also utilized to confirm the activity of the wild-type, C323S, C266A, C266S, and C266S/C323S enzymes.

ESR Methodology for Structural Characterization of HMG-CoA Lyase Mutants

Measurement of Mn binding to human HMG-CoA lyase by ESR was performed as follows. X-band ESR spectra were recorded using a Varian Century-Line 9-GHz spectrometer with a TE cavity at 22 °C and a modulation amplitude of 10 G, modulation frequency of 100 KHz, and microwave power of 60 milliwatts. The field sweep was 1000 G, and the time constant was 0.25 s. A quartz microflat cell was used for all measurements.

Human lyase samples in 10 mM potassium phosphate, pH 7.8, containing 100 mM NaCl, 20% glycerol, and 1.0 mM dithiothreitol were concentrated to 100-150 µM (calculated on the basis of a 34-kDa subunit) using Amicon Centriflo membrane cones. Glycerol was removed from the samples prior to ESR experiments using Sephadex G-50 centrifugal columns(22) . The ESR samples contained variable concentrations of Mn (25-182 µM) with either 100 µM (wild-type) or 68 µM (mutant) HMG-CoA lyase sites. Mn bound to HMG-CoA lyase was calculated by comparing the amplitudes of the sample spectra with the corresponding amplitudes observed with a solution containing an equal concentration of Mn in buffer (10 mM potassium phosphate, 100 mM NaCl, 1.0 mM dithiothreitol, pH 7.8). The data were subjected to Scatchard analysis; data plots were fit using linear regression analysis.


RESULTS

Rationale for Mutagenesis of Cysteine 266

Hruz et al.(11) were able to identify Cys-237 of the bacterial enzyme as a residue potentially important for catalysis. The stoichiometry of the affinity label 2-butynoyl-CoA bound to the recombinant human enzyme (1.0 per subunit(10) ) paralleled the results determined for both the bacterial and avian lyases (0.9 and 0.8 per subunit(11) ). These observations are most simply explained by the existence of an equivalent target in all three enzymes. Sequence homology suggests that Cys-266 of the human lyase corresponds to Cys-237 of the bacterial enzyme (Table 1).



Additional rationale for testing the function of a cysteine within the substrate-binding site stems from the pH dependence of the HMG-CoA lyase reaction that has been reported for purified avian liver lyase (19) and for the partially purified bovine liver (18) and bacterial (23) enzymes. In all cases, the pH optimum is distinctly alkaline, but no pK estimates were offered in those earlier reports nor were details provided to indicate how closely the activity estimates approximate V. The pH dependence of V for both C323S (Fig. 1A) and wild-type (data not shown) human HMG-CoA lyases was found to be identical. The curve shown fit to the data was calculated for ionization of a single group with a pK value of 7.99 ± 0.06. Such observations for C323S lyase eliminate any possibility that the regulatory sulfhydryl group of Cys-323 is responsible for the observed pH profile. However, it remains possible that the observed pH dependence reflects the requirement to form a thiolate anion from a different cysteine located in the active site.


Figure 1: pH dependence of human HMG-CoA lyase activity. , measurements in 100 mM Tris-HCl, 0.1 mM EDTA; , measurements in 100 mM glycine, 0.1 mM EDTA buffer. A, for C323S lyase, reaction rates were measured by the enzymatic spectrophotometric coupled assay as described under ``Experimental Procedures.'' The pH/rate profile was fit using a curve calculated for ionization of a group with a pK value of 7.99 ± 0.06. B, for C266A/C323S HMG-CoA lyase, reaction rates were measured by the radioactive assay as described under ``Experimental Procedures.'' Linear regression analysis was used to generate the line shown fit to the data.



The utility of a bacterial expression system for production of recombinant human HMG-CoA lyase has already been demonstrated in mutagenesis work that produced an active, oxidation-resistant C323S lyase variant(10) . To test the hypothesis that Cys-266 plays an important role in catalysis, the Cys-266 sulfhydryl was eliminated by synthetic oligonucleotide cassette mutagenesis. Substitutions were engineered in both pTrcHL-1 and pTrcHL-C323S expression plasmids. The conservative substitutions correspond to replacement of the cysteine sulfhydryl with either a serine hydroxyl (C266S, C266S/C323S) or the C-3 proton of an alanine (C266A, C266A/C323S).

Kinetic Characterization of Human HMG-CoA Lyase Mutants

Utilizing the purification system developed earlier for human HMG-CoA lyase(10) , each of the Cys-266 mutant constructs was expressed and purified. All Cys-266 variants of HMG-CoA lyase were found in the soluble bacterial extracts at levels similar to those observed upon expression of both wild-type and C323S enzymes. Each mutant lyase purified identically to the wild-type enzyme; the elution profiles for the anion-exchange, hydrophobic interaction, and gel filtration chromatographic steps for each of the Cys-266 constructs were identical to those observed with the wild-type enzyme, suggesting that the overall conformation of each mutant is not drastically altered. Each variant was purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis (data not shown). The apparent subunit molecular weight (M = 34,000) was identical to that observed for both wild-type and C323S enzymes.

The specific activity of the purified Cys-266 mutants is summarized in Table 2. The serine-containing variants (C266S and C266S/C323S) exhibited 700-1300-fold diminution in activity. In the case of the alanine mutants (C266A and C266A/C323S), the diminution in activity in comparison with wild-type (or C323S) lyase was even more prominent, with a net effect of >10-fold. In contrast to the 3-4 orders of magnitude difference in catalytic efficiency between the serine or alanine mutants and the wild-type enzyme, substantial differences in K values for HMG-CoA were not apparent (Table 2), suggesting that the active site is not grossly altered when Cys-266 is replaced.



The pH dependence of the C266A/C323S variant, which exhibits the greatest diminution in catalytic function, was measured utilizing the radioactive [C]HMG-CoA depletion assay. Qualitatively, the data (Fig. 1B) indicate an increase in rate as pH rises from values of 8 to 10. Despite the increased sensitivity of this assay, signal-to-noise considerations (at lower radioactive substrate levels) precluded a full substrate concentration dependence study at pH <8.2. In such cases, the observed rate was unchanged when measured at several elevated substrate levels, suggesting that these rate estimates approach V. The data for this mutant enzyme could not be satisfactorily fit to a single ionization curve by the algorithm used to generate the theoretical fit shown in Fig. 1A for the catalytically functional C323S lyase. Interpretation is complicated by the fact that, at low pH, no deviation from the linear relationship between V and pH could be detected before activity diminished to levels where signal-to-noise limitations precluded meaningful rate estimates. At elevated pH, activity increased linearly with pH increment until further estimates were precluded by enzyme denaturation due to alkalinity of the assay mixture.

Characterization of the Structural Integrity of HMG-CoA Lyase Mutants

To test whether the marked diminution in catalytic activity observed for Cys-266 mutants may be the consequence of a structure that is seriously perturbed in comparison with that of active wild-type or C323S HMG-CoA lyase, biophysical characterization of these mutants was performed. CD spectroscopy (data not shown) did not reveal significant differences between catalytically active wild-type or C323S lyase and the Cys-266 mutant proteins, suggesting that secondary structure is not substantially different in these proteins. However, it remained possible that more subtle perturbations than can be detected by this optical spectroscopy approach could account for the observed diminution in activity. This concern prompted development of a more refined approach to test this issue.

Activities of wild-type and C323S human lyases(10) , avian lyase(24) , and bacterial lyase (9) are all markedly stimulated by divalent cations. The activity of each Cys-266 variant was also found to be dependent on the presence of a divalent cation. Wild-type and C323S human lyases exhibit K values for Mg that are 100-fold weaker than reported for the Kvalues for Mn(10) . When the cation concentration dependence data, measured under conditions of saturating HMG-CoA, are extrapolated for estimation of V, equivalent values are observed using either Mg or Mn, underscoring the efficacy of Mn as an activator of the human enzyme. The observation that Mn, which is spectroscopically active due to its five unpaired d electrons, binds with such high affinity in comparison with the spectroscopically silent cation Mg suggested the utility of this cation as a structural probe for the enzyme and its variants. For this reason, the binding of manganese to the enzyme was investigated by both kinetic and ESR spectroscopic methods.

The K value previously reported (10) for Mn (0.34 µM) was determined under saturating HMG-CoA conditions. To determine the activator constant (K) for Mn, the activity of HMG-CoA lyase was determined using variable concentrations of both Mn and HMG-CoA; Fig. 2shows a double-reciprocal plot of these data. Based on the intersection point of the lines(25) , an activator constant, which represents an estimate of an equilibrium dissociation constant (26) for the binary Mn-lyase complex, was calculated for human HMG-CoA lyase (K = 0.5 µM).


Figure 2: Double-reciprocal plot of the initial velocity of human HMG-CoA lyase as a function of Mn concentration. Concentrations of the substrate, HMG-CoA, were 5 (), 10 (), 25 (), 50 (), and 100 () µM. The activator constant (K) was calculated from the intersection point.



The binding of activator Mn to human HMG-CoA lyase was directly measured by ESR spectroscopy. Before conducting these measurements, the stability of HMG-CoA lyase in the absence of glycerol had to be determined. The viscosity that results from inclusion of a significant concentration of glycerol in the sample can lead to broadening of the Mn signal, complicating interpretation of the resulting data. This constraint seemed potentially serious since bacterial HMG-CoA lyase loses activity rapidly in the absence of glycerol(9) . To evaluate the consequences for the human enzyme, glycerol was rapidly removed from both the wild-type and C323S lyases by centrifugal gel filtration. Both glycerol-free samples were incubated at 4 °C, and the activity was periodically measured under standard assay conditions. The half-life of the wild-type enzyme was determined to be >12 h, which is sufficient for execution of extensive ESR experiments prior to any significant decrease in activity. Unfortunately, the half-life of C323S was <4 h. In this respect, C323S human lyase was very similar to bacterial HMG-CoA lyase, which also lacks the C-terminal region cysteine residue.

Since the C323S variants were not stable for extended periods of time after the removal of glycerol, the Mn ESR experiments were performed with either the wild-type enzyme or the Cys-266 mutants that, like wild-type lyase, contained Cys-323. The spectrum of free Mn (90 µM) is shown as the larger amplitude trace in the inset of Fig. 3. When wild-type HMG-CoA lyase (100 µM sites) was added, the free Mn was diminished upon binding of this activator cation by the enzyme, and the amplitude of the signal was correspondingly decreased (Fig. 3, inset). By comparing the peak-to-peak amplitudes of corresponding lines of Mn spectra measured in the absence and presence of lyase, the amount of bound manganese was determined. While signal-to-noise considerations limit accurate measurements to mixtures with total Mn >25 µM, the data are adequate for Scatchard analysis (Fig. 3), which provides a good estimate of the binding stoichiometry (n = 0.7). Linear regression analysis of the data also provides a reasonable slope estimate, used for calculation of a dissociation constant (K = 1.5 µM) for the binary EM complex (Table 2). The K value determined by these physical measurements is in good agreement with the kinetically determined activator constant (K = 0.5 µM). When similar experiments were performed using C266S and C266A enzymes, Scatchard analyses indicated K values of 7.9 and 22.6 µM, respectively (Table 2). While these values indicate slightly weaker binding than measured with wild-type lyase, differences are modest in comparison with the observed contrasts in catalytic efficiency. Significantly, the stoichiometry of activator Mn binding, which can be well determined in the samples due to the high Mn site occupancy, is comparable for both the wild-type enzyme and the Cys-266 variants (Table 2), indicating that these mutants possess a full complement of functional binding sites. Therefore, these data confirm that the active-site structure of the Cys-266 variants is not grossly altered by the conservative substitution of serine or alanine.


Figure 3: Mn binding to HMG-CoA lyase. Enzyme was freed of glycerol as described under ``Experimental Procedures'' and maintained on ice until used for sample preparation. Approximately 10 min were required for sample equilibration at 22 °C and subsequent ESR measurement; no significant loss of enzyme activity occurs in this time period. Inset, spectra of Mn (90 µM) in the absence or presence of HMG-CoA lyase (100 µM). Each xaxis division corresponds to 100 G. Instrumental conditions were as follows: modulation amplitude, 10 G; modulation frequency, 100 KHz; microwave power, 60 milliwatts; center field, 3000 G; field sweep, 1000 G; scan time, 4 min; and time constant, 0.25 s. Main panel, Scatchard plot of the ESR data for Mn binding to wild-type human HMG-CoA lyase. By comparing the amplitudes of the Mn ESR spectra in the absence and presence of HMG-CoA lyase, the amount of bound Mn was determined. Linear regression analysis was used to calculate the line shown fit to the data.




DISCUSSION

Engineering mutations into HMG-CoA lyase has proven informative in the context of evaluating regulatory (10) and catalytic (this report) regions within the enzyme. Such an approach may also prove useful as we model the point mutations associated with defects in this protein that produce hydroxymethylglutaric aciduria in humans. For this reason, attempts were made to develop methods useful for evaluation of the structural integrity of mutant human HMG-CoA lyase proteins.

Previous work with both bacterial HMG-CoA lyase (27) and avian HMG-CoA synthase (13) has demonstrated the spin-labeled substrate analog RCoA (carboxy-PROXYL-CoA(28) ) to be a useful probe in ESR characterization of wild-type and mutant forms of these enzymes. This analog not only proves to be a competitive inhibitor with respect to acyl-CoA substrate, but also can be shown by physical measurements to bind to the active site of both enzymes with Kvalues comparable to the kinetically determined Kestimates. When the utility of this spin probe was evaluated using human HMG-CoA lyase, inhibition (K > 500 µM) was observed at levels of RCoA that are severalfold higher than used with the bacterial lyase (K = 98 µM), and therefore, this analog is unattractive for ESR spectroscopy studies. Differences between bacterial and human enzymes with respect to RCoA binding are not surprising since binding of 2-butynoyl-CoA to the avian enzyme was 5-fold weaker than to the bacterial enzyme(11) . On the basis of these results, the dissociable divalent cation activator was considered for use as a physical probe.

While the constraint that ESR measurements of Mn binding required elimination of glycerol from the enzyme buffer restricted such measurements to wild-type, C266S, and C266A lyases, the value of Mn as a structural probe has been amply demonstrated. The observation that the mutant lyases possess a full complement of high affinity binding sites for activator cation represents a stringent criterion of structural integrity that complements secondary structure comparisons. An additional benefit of the ESR approach to quantitate cation affinity involves the consequent refinement of our understanding of the mechanism of cation activation of HMG-CoA cleavage. Demonstration by the biophysical approach that a binary Mn-enzyme complex forms with a Kvalue comparable to the kinetically determined Kvalue argues that lyase functions as a metal-enzyme complex. This direct measurement allows us to refine earlier speculation, based on indirect kinetic data generated using an alternative substrate(24) , by now suggesting that formation of a binary enzyme-cation species is a prelude to production of a catalytically active ternary enzyme-substrate-metal complex.

Finally, the marked diminution in catalytic activity that coincides with replacement of the Cys-266 sulfhydryl in the otherwise structurally intact HMG-CoA lyase mutants invites comment. The enzyme catalyzes a typical Claisen cleavage. Hanson and Rose (29) have proposed that enzymatic reactions of this type involve general acid and general base catalysts oriented on opposite sides of the substrate. The pH/rate profile for wild-type human lyase is compatible with the hypothesis that deprotonation of an amino acid side chain exhibiting a pK value of 8.0 coincides with formation of a catalytically functional enzyme. Is it plausible that the data reflect ionization of Cys-266 and that this residue functions as the general base? Replacement of the Cys-266 sulfhydryl with a higher pK serine hydroxyl lowers activity by 3 orders of magnitude. The effect increases to 4 orders of magnitude in the C266A and C266A/C323S mutants, which is compatible with the elimination of any nucleophile at this position. The magnitude of these effects precludes performing rate measurements at pH 7 just as alkaline lability of the protein precludes measurements at pH 10. With these constraints, it is unclear whether the increase in rate for C266A as pH rises reflects titration of an amino acid side chain other than the Cys-266 sulfhydryl or whether specific base catalysis by solvent-derived hydroxyl ion accounts for the effect. In the related lyase reaction catalyzed by Escherichia coli phospho-2-keto-3-deoxyheptulonate aldolase (EC 4.1.2.15; 3-deoxyarabinoheptulosonate-7-phosphate synthase), mutagenic replacement of the Cys-61 sulfhydryl resulted in diminution of activity by 10-fold(30) , an effect comparable in magnitude to our observation with C266A lyase mutants. The cysteine residue was viewed as critical to catalysis, but was assigned a role in metal binding. In the case of rabbit muscle fructose-1,6-bisphosphate aldolase (EC 4.1.2.13), a more exaggerated effect (10-fold diminution in activity) results when the -amino group of Lys-146 is eliminated(31) . Only a 10-fold drop in activity is observed when histidine is substituted for lysine at this position. Potential roles proposed for this lysine include stabilization of a carbanionic reaction intermediate, electrostatic interaction with an acidic active-site residue to enhance that residue's function as a catalytic base, and a direct general base function for lysine's -amino group in accepting a proton from the substrate's C-4 hydroxyl group. This latter possibility would appear analogous to the requirement for a general base to accept a proton from the C-3 hydroxyl of the substrate in the HMG-CoA lyase reaction. The retention of measurable activity in the Cys-266 HMG-CoA lyase mutants suggests that an unambiguous assignment of this residue as a catalytic base cannot yet be made. As in the case of aldolase, an alternative role in enhancing the reactivity of another active-site amino acid that participates more directly in reaction chemistry remains to be excluded. Nonetheless, the 10-fold effect observed upon eliminating the Cys-266 sulfhydryl of HMG-CoA lyase certainly qualifies Cys-266 as a catalytic residue, validating the active-site assignment made on the basis of protein chemistry studies(11) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK21491 and March of Dimes Birth Defects Foundation Basic Research Grants FY93-0234 and FY94-0880 (to H. M. M.) and National Institutes of Health National Research Service Award DK09018 (to J. R. R.). ESR measurements were performed using the facilities of the National Biomedical ESR Center, Medical College of Wisconsin, supported by National Institutes of Health Grant RR-01008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
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: 414-266-8497.

The abbreviation used is: HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA.

The numbering convention designates as residue 1 the first residue in the precursor form of this mitochondrial matrix enzyme.


ACKNOWLEDGEMENTS

Alison Carstens expertly expressed and purified the HMG-CoA lyase enzymes used in these studies. Grant Mitchell (Hopital Ste. Justine, University of Montreal) provided plasmid pETHL-1, which contains the human HMG-CoA lyase-encoding cDNA that was used to construct the expression plasmids used in this study. Liane Mende-Mueller (Medical College of Wisconsin Protein/Nucleic Acid Facility) supervised production of the oligonucleotides used for cassette mutagenesis.


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