FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2071581231> ?p ?o ?g. }

• W2071581231 endingPage "17953" @default.
• W2071581231 startingPage "17946" @default.
• W2071581231 abstract "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. 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. 3-hydroxy-3-methylglutaryl-CoA 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl-CoA polymerase chain reaction electron spin resonance 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)1 synthase catalyzes the committed step in ketogenic and cholesterogenic pathways. Early work (1.Miziorko H.M. Clinkenbeard K.D. Reed W.D. Lane M.D. J. Biol. Chem. 1975; 250: 5768-5773Abstract Full Text PDF PubMed Google Scholar, 2.Miziorko H.M. Lane M.D. J. Biol. Chem. 1977; 252: 1414-1420Abstract Full Text PDF PubMed Google Scholar) on the purified enzyme identified reaction intermediates that indicate that catalysis of HMG-CoA production involves a three-step process (Scheme FS1). Recombinant forms of both the avian (3.Misra I. Narasimhan C. Miziorko H.M. J. Biol. Chem. 1993; 268: 12129-12136Abstract Full Text PDF PubMed Google Scholar) and human (4.Rokosz L.L. Boulton D.A. Butkiewicz E.A. Sanyal G. Cueto M.A. Lachance P.A. Hermes J.D. Arch. Biochem. Biophys. 1994; 312: 1-13Crossref PubMed Scopus (42) Google Scholar) 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.Rokosz L.L. Boulton D.A. Butkiewicz E.A. Sanyal G. Cueto M.A. Lachance P.A. Hermes J.D. Arch. Biochem. Biophys. 1994; 312: 1-13Crossref PubMed Scopus (42) Google Scholar). Mutagenesis work on the recombinant avian enzyme (3.Misra I. Narasimhan C. Miziorko H.M. J. Biol. Chem. 1993; 268: 12129-12136Abstract Full Text PDF PubMed Google Scholar) has confirmed that Cys129 is required to form the acetyl-S-enzyme intermediate (Scheme FS1), a hypothesis that was initially advanced on the basis of protein modification by a mechanism-based inhibitor (5.Miziorko H.M. Behnke C.E. Biochemistry. 1985; 24: 3174-3179Crossref PubMed Scopus (33) Google Scholar, 6.Miziorko H.M. Behnke C.E. J. Biol. Chem. 1985; 260: 13513-13516Abstract Full Text PDF PubMed Google Scholar) as well as sequence analysis of a peptide that harbors the acetyl-S-Cys129 adduct (7.Vollmer S.H. Mende-Mueller L.M. Miziorko H.M. Biochemistry. 1988; 27: 4288-4292Crossref PubMed Scopus (24) Google Scholar). 2The residue numbering convention follows the sequence of the avian and human cytosolic proteins. Additionally, kinetic characterization of mutants in which His264 has been replaced implicates that residue (8.Misra I. Miziorko H.M. Biochemistry. 1996; 35: 9610-9616Crossref PubMed Scopus (28) Google Scholar) 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.Chun K.Y. Miziorko H.M. FASEB J. 1998; 12 (abstr.): 1359Crossref PubMed Scopus (111) Google Scholar), 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. Escherichia coli BL21 (DE3) and the expression vector pET-3d were purchased from Novagen (Madison, WI). E. colistrain DH5α 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. PfuDNA 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-β-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. 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). Point mutations were engineered in HMG-CoA synthase encoding cDNA by using the overlap extension PCR technique (10.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). 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 DH5α 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. The procedure (3.Misra I. Narasimhan C. Miziorko H.M. J. Biol. Chem. 1993; 268: 12129-12136Abstract Full Text PDF PubMed Google Scholar) 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.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar), using bovine serum albumin as the standard. The purity of the enzymes was assessed by SDS-polyacrylamide gel electrophoresis. Synthesis was performed according to Miziorko and Behnke (5.Miziorko H.M. Behnke C.E. Biochemistry. 1985; 24: 3174-3179Crossref PubMed Scopus (33) Google Scholar). 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. Due to the low catalytic activity of E95A synthase, a standard spectrophotometric assay (12.Clinkenbeard K.D. Reed W.D. Mooney R.A. Lane M.D. J. Biol. Chem. 1975; 250: 3108-3116Abstract Full Text PDF PubMed Google Scholar) could not be employed to obtain initial velocity data. The equivalent radioisotopic assay (12.Clinkenbeard K.D. Reed W.D. Mooney R.A. Lane M.D. J. Biol. Chem. 1975; 250: 3108-3116Abstract Full Text PDF PubMed Google Scholar) 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 mmsodium 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.Vollmer S.H. Mende-Mueller L.M. Miziorko H.M. Biochemistry. 1988; 27: 4288-4292Crossref PubMed Scopus (24) Google Scholar). 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.Miziorko H.M. Clinkenbeard K.D. Reed W.D. Lane M.D. J. Biol. Chem. 1975; 250: 5768-5773Abstract Full Text PDF PubMed Google Scholar). 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 mmHCl 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.Miziorko H.M. Clinkenbeard K.D. Reed W.D. Lane M.D. J. Biol. Chem. 1975; 250: 5768-5773Abstract Full Text PDF PubMed Google Scholar) 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. 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. 1H, 13C (ω) 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 (δ = 0 ppm). Residual HOD resonance was suppressed by lower-power presaturation.13C decoupling in ω2 was carried out using the GARP decoupling scheme. The spectra were obtained using a1H 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 of13C-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 the1H 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. 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.Alter G.M. Casazza J.P. Zhi W. Nemeth P. Srere P.A. Evans C.T. Biochemistry. 1990; 29: 7557-7563Crossref PubMed Scopus (69) Google Scholar) and fructose 1,6-bisphosphate aldolase (14.Morris A.J. Tolan D.R. J. Biol. Chem. 1993; 268: 1095-1100Abstract Full Text PDF PubMed Google Scholar), 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. 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.Misra I. Narasimhan C. Miziorko H.M. J. Biol. Chem. 1993; 268: 12129-12136Abstract Full Text PDF PubMed Google Scholar); the mutant enzyme displays identical chromatographic properties and exhibits a comparable level of homogeneity (Fig. 2) and stability. 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.Misra I. Narasimhan C. Miziorko H.M. J. Biol. Chem. 1993; 268: 12129-12136Abstract Full Text PDF PubMed Google Scholar, 15.Vinarov D.A. Narasimhan C. Miziorko H.M. J. Am. Chem. Soc. 1999; 121: 270-271Crossref Scopus (9) Google Scholar) 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.Table IKinetic constants, physical parameters, and binding properties of E95A HMG-CoA synthaseParameterWild type HMG-CoA synthaseE95A HMG-CoA synthaseV max, overall reaction (units/mg)aThe rate of the overall reaction was determined by radioisotopic assay. Rates were measured in the presence of a saturating level of acetoacetyl-CoA and variable concentrations of acetyl-CoA.4.4 ± 0.39<0.00002K m of acetyl-CoA (μm)290 ± 22NDbND, not determined; activity was too low to allow accurate determination.K m of acetoacetyl-CoA (μm)cK m of AcAc-CoA is an apparent value, determined in the presence of 200 μm acetyl-CoA.1.2 ± 0.12NDStoichiometry of R · CoA bindingdDissociation constant, binding stoichiometry, and rotational correlation time for R · CoA were calculated for wild type and E95A synthase enzymes as described under “Experimental Procedures.” Stoichiometry is calculated based on a 57.6-kDa subunit.0.90 ± 0.180.95 ± 0.11K d for R · CoA (μm)dDissociation constant, binding stoichiometry, and rotational correlation time for R · CoA were calculated for wild type and E95A synthase enzymes as described under “Experimental Procedures.” Stoichiometry is calculated based on a 57.6-kDa subunit.102 ± 2028 ± 2Rotational correlation time, τc (ns)dDissociation constant, binding stoichiometry, and rotational correlation time for R · CoA were calculated for wild type and E95A synthase enzymes as described under “Experimental Procedures.” Stoichiometry is calculated based on a 57.6-kDa subunit.35 ± 435 ± 413C chemical shifts for [1,2-13C]acetyl-S-enzyme (ppm)eSample preparation and NMR parameters are discussed under “Experimental Procedures.” C-1184.25184.18 C-225.8225.79a The rate of the overall reaction was determined by radioisotopic assay. Rates were measured in the presence of a saturating level of acetoacetyl-CoA and variable concentrations of acetyl-CoA.b ND, not determined; activity was too low to allow accurate determination.c K m of AcAc-CoA is an apparent value, determined in the presence of 200 μm acetyl-CoA.d Dissociation constant, binding stoichiometry, and rotational correlation time for R · CoA were calculated for wild type and E95A synthase enzymes as described under “Experimental Procedures.” Stoichiometry is calculated based on a 57.6-kDa subunit.e Sample preparation and NMR parameters are discussed under “Experimental Procedures.” Open table in a new tab 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 andK 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.McCalley R.C. Shimshick E.J. McConnell H.M. Chem. Phys. Lett. 1972; 13: 115Crossref Scopus (200) Google Scholar). This group remains substantially immobilized when R·CoA binds to E95A. The estimate of correlation time (τc = 35 ns; Table I) is indistinguishable from the value reported for wild type enzyme (3.Misra I. Narasimhan C. Miziorko H.M. J. Biol. Chem. 1993; 268: 12129-12136Abstract Full Text PDF PubMed Google Scholar, 17.Miziorko H.M. Lane M.D. Weidman S.W. Biochemistry. 1979; 18: 399-403Crossref PubMed Scopus (11) Google Scholar). 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, theoverall tertiary structure is also intact. 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. Vinarovet al. (15.Vinarov D.A. Narasimhan C. Miziorko H.M. J. Am. Chem. Soc. 1999; 121: 270-271Crossref Scopus (9) Google Scholar) 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. As indicated in Scheme FS1, 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.Vollmer S.H. Mende-Mueller L.M. Miziorko H.M. Biochemistry. 1988; 27: 4288-4292Crossref PubMed Scopus (24) Google Scholar) 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 the13C 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 biophysicalargument that these species are functionally equivalent. Thechemical 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.Table IIPartial reactions catalyzed by E95A HMG-CoA synthaseParameterWild type HMG-CoA synthaseE95AV max, acetyl-CoA hydrolysis (units/mg)aHydrolysis activity was measured by converting residual [14C]acetyl-CoA to acid-stable [14C]citrate with excess citrate synthase and oxaloacetate.0.016 ± 0.0030.010 ± 0.001K m of acetyl-CoA, acetyl-CoA hydrolysis (μm)aHydrolysis activity was mea" @default.
• W2071581231 created "2016-06-24" @default.
• W2071581231 creator A5060805525 @default.
• W2071581231 creator A5064542915 @default.
• W2071581231 creator A5073168915 @default.
• W2071581231 creator A5074318077 @default.
• W2071581231 date "2000-06-01" @default.
• W2071581231 modified "2023-09-30" @default.
• W2071581231 title "3-Hydroxy-3-methylglutaryl-CoA Synthase" @default.
• W2071581231 cites W1513122208 @default.
• W2071581231 cites W1534425037 @default.
• W2071581231 cites W1546419331 @default.
• W2071581231 cites W1556878161 @default.
• W2071581231 cites W1572303377 @default.
• W2071581231 cites W1585817752 @default.
• W2071581231 cites W1684142465 @default.
• W2071581231 cites W1875092895 @default.
• W2071581231 cites W1971661218 @default.
• W2071581231 cites W1977348707 @default.
• W2071581231 cites W1987314407 @default.
• W2071581231 cites W1991362153 @default.
• W2071581231 cites W1994116487 @default.
• W2071581231 cites W1998593869 @default.
• W2071581231 cites W2000247137 @default.
• W2071581231 cites W2002402206 @default.
• W2071581231 cites W2031487936 @default.
• W2071581231 cites W2042815508 @default.
• W2071581231 cites W2052539013 @default.
• W2071581231 cites W2060450389 @default.
• W2071581231 cites W2060913542 @default.
• W2071581231 cites W2080723504 @default.
• W2071581231 cites W2091201743 @default.
• W2071581231 cites W2923667469 @default.
• W2071581231 cites W4293247451 @default.
• W2071581231 cites W4322701616 @default.
• W2071581231 doi "https://doi.org/10.1074/jbc.m909725199" @default.
• W2071581231 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10748155" @default.
• W2071581231 hasPublicationYear "2000" @default.
• W2071581231 type Work @default.
• W2071581231 sameAs 2071581231 @default.
• W2071581231 citedByCount "28" @default.
• W2071581231 countsByYear W20715812312012 @default.
• W2071581231 countsByYear W20715812312013 @default.
• W2071581231 countsByYear W20715812312014 @default.
• W2071581231 countsByYear W20715812312016 @default.
• W2071581231 countsByYear W20715812312017 @default.
• W2071581231 countsByYear W20715812312019 @default.
• W2071581231 countsByYear W20715812312020 @default.
• W2071581231 countsByYear W20715812312021 @default.
• W2071581231 countsByYear W20715812312022 @default.
• W2071581231 crossrefType "journal-article" @default.
• W2071581231 hasAuthorship W2071581231A5060805525 @default.
• W2071581231 hasAuthorship W2071581231A5064542915 @default.
• W2071581231 hasAuthorship W2071581231A5073168915 @default.
• W2071581231 hasAuthorship W2071581231A5074318077 @default.
• W2071581231 hasBestOaLocation W20715812311 @default.
• W2071581231 hasConcept C112243037 @default.
• W2071581231 hasConcept C181199279 @default.
• W2071581231 hasConcept C185592680 @default.
• W2071581231 hasConcept C55493867 @default.
• W2071581231 hasConceptScore W2071581231C112243037 @default.
• W2071581231 hasConceptScore W2071581231C181199279 @default.
• W2071581231 hasConceptScore W2071581231C185592680 @default.
• W2071581231 hasConceptScore W2071581231C55493867 @default.
• W2071581231 hasIssue "24" @default.
• W2071581231 hasLocation W20715812311 @default.
• W2071581231 hasOpenAccess W2071581231 @default.
• W2071581231 hasPrimaryLocation W20715812311 @default.
• W2071581231 hasRelatedWork W1569138873 @default.
• W2071581231 hasRelatedWork W2008886368 @default.
• W2071581231 hasRelatedWork W2015818636 @default.
• W2071581231 hasRelatedWork W2052443581 @default.
• W2071581231 hasRelatedWork W2062914927 @default.
• W2071581231 hasRelatedWork W2067065606 @default.
• W2071581231 hasRelatedWork W2095027488 @default.
• W2071581231 hasRelatedWork W2095865899 @default.
• W2071581231 hasRelatedWork W284590271 @default.
• W2071581231 hasRelatedWork W1588549711 @default.
• W2071581231 hasVolume "275" @default.
• W2071581231 isParatext "false" @default.
• W2071581231 isRetracted "false" @default.
• W2071581231 magId "2071581231" @default.
• W2071581231 workType "article" @default.