Human Cytoplasmic 3-Hydroxy-3-methylglutaryl Coenzyme A Synthase: Expression, Purification, and Characterization of Recombinant Wild-Type and Cys129 Mutant Enzymes

Rokosz, Laura L.; Boulton, D. A.; Butkiewicz, E. A.; Sanyal, Gautam; Cueto, Maria; Lachance, Paul; Hermes, Jeff

doi:10.1006/abbi.1994.1273

Cited by 51 publications

(47 citation statements)

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“…A time-dependent loss of activity was observed; inactivation occurred with first-order kinetics, as expected for covalent modification. Computational fits of these data indicate an affinity for hymeglusin (K i ϭ 570 Ϯ 120 nM; Table 1) that is over an order of magnitude weaker than the value (53.7 nM) reported for human HMGCS (23). However, the rate of inactivation (k inact ϭ 17 Ϯ 3 min Ϫ1 ) is an order of magnitude greater than that observed for the human enzyme (1.06 min Ϫ1 ).…”

Section: Fig 2 Hmgcs Multiple-sequence Alignment the Sequence Of Hvhmentioning

confidence: 81%

“…The very small difference between the K m and K i values for acetoacetyl-CoA (0.5 and 1.9 M, respectively; Table 1) leads to a sharp departure of the rate-versus-concentration curve from the hyperbolic shape observed for HvHMGCS where there is no strong substrate inhibition. For eukaryotic HMGCS (e.g., the yeast [26] and human [23] enzymes), substrate inhibition by acetoacetyl-CoA has also been reported but more gradual rate decreases occur at elevated substrate concentrations. Estimates of their K i values for acetoacetyl-CoA are lower in affinity (8 and 12 M, respectively).…”

Section: Discussionmentioning

confidence: 99%

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Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

VanNice¹,

Skaff²,

Wyckoff³

et al. 2013

Journal of Bacteriology

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Enzymes of the isoprenoid biosynthetic pathway in halophilic archaea remain poorly characterized, and parts of the pathway remain cryptic. This situation may be explained, in part, by the difficulty of expressing active, functional recombinant forms of these enzymes. The use of newly available expression plasmids and hosts has allowed the expression and isolation of catalytically active Haloferax volcanii 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (EC 2.3.310). This accomplishment has permitted studies that represent, to the best of our knowledge, the first characterization of an archaeal hydroxymethylglutaryl CoA synthase. Kinetic characterization indicates that, under optimal assay conditions, which include 4 M KCl, the enzyme exhibits catalytic efficiency and substrate saturation at metabolite levels comparable to those reported for the enzyme from nonhalophilic organisms. This enzyme is unique in that it is the first hydroxymethylglutaryl CoA synthase that is insensitive to feedback substrate inhibition by acetoacetyl-CoA. The enzyme supports reaction catalysis in the presence of various organic solvents. Haloferax 3-hydroxy-3-methylglutaryl CoA synthase is sensitive to inactivation by hymeglusin, a specific inhibitor known to affect prokaryotic and eukaryotic forms of the enzyme, with experimentally determined K i and k inact values of 570 ؎ 120 nM and 17 ؎ 3 min ؊1 , respectively. In in vivo experiments, hymeglusin blocks the propagation of H. volcanii cells, indicating the critical role that the mevalonate pathway plays in isoprenoid biosynthesis by these archaea

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Section: Fig 2 Hmgcs Multiple-sequence Alignment the Sequence Of Hvhmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

VanNice¹,

Skaff²,

Wyckoff³

et al. 2013

Journal of Bacteriology

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“…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][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetyl-CoA was purchased from Moravek Biochemicals (Brea, CA) and 3-chloro- [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]propionic acid from American Radiolabeled Chemicals (St. Louis, MO). All other reagents were purchased from Sigma, Aldrich, Amersham Pharmacia Biotech, or Bio-Rad.…”

Section: Methodsmentioning

confidence: 99%

“…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 Cys 129 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-Cys 129 adduct (7).…”

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confidence: 99%

3-Hydroxy-3-methylglutaryl-CoA Synthase

Chun¹,

Vinarov²,

Zajíček³

et al. 2000

Journal of Biological Chemistry

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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 acylCoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by 13 C 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 Glu 95 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 Glu 95 as required to support its function as a general acid.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 HMGCoA production involves a three-step process (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 Cys 129 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-Cys 129 adduct (7).2 Additionally, kinetic characterization of mutants in which His 264 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 Glu 95 as a residue that is important to reaction chemistry. This report documents the crucial role of Glu 95 in catalysis and indicates which of the three steps in the reaction is compromised upon replacement of the Glu 95 carboxyl. Finally, potential functions for Glu 95 are considered, and tests ai...

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“…Indeed, a cysteine residue at the active site, which is involved in covalent thioester formation with the substrate acetyl-CoA in the first step of the catalytic cycle, has been strongly implicated in the yeast protein (26)(27)(28) and demonstrated to be present in the avian liver (29-31) and human cytoplasmic (22) enzymes. While these results are not surprising, it is interesting to note that cerulenin has been shown to inhibit the yeast enzyme (32), but not that of avian liver (5).…”

Section: Resultsmentioning

confidence: 99%

Irreversible inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from yeast by F-244 and (RS)-β-butyrolactone

Bell¹,

Saepudin²,

Harrison³

1996

Can. J. Chem.

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The inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase from yeast was compared for both F-244 (1) and (RS)-P-butyrolactone (5). F-244 exhibits irreversible inhibition with an IC,, of 8 nM, similar to that reported for the rat liver enzyme, while the binding constant (IIK,) and inactivation rate constant (kill,,,) are similar to values reported for the human cytoplasmic enzyme. (RS)-P-Butyrolactone (5) also irreversibly inhibits the enzyme, but with much lower efficiency (IC,, 2 mM). The values for K, (9 mM) and kill,,, (0.0078 s-I) for 5 were determined. The results show that kin,,, for 5 and 1 differ by a factor of only 2.5, while KI for 5 is higher by a factor of 1.8 x 10,. Hence, the p-lactone ring is shown to be the sole essential structural feature in 1 for irreversible inactivation of HMG-CoA synthase; however, the remaining functionality enhances the binding of 1 to the enzyme relative to 5.Key words: HMG-CoA synthase, F-244, butyrolactone, irreversible, inhibitor.RCsumC : On a compart l'inhibition de la synthase 3-hydroxy-3-mtthylglutaryl coenzyme A (HMG-CoA) vis-a-vis le compost F-244 (1) anisi que de la (RS)-P-butyrolactone (5). Le composk F-244 prtsente une inhibition irrtversible avec un IC5, de 8 nM, semblable B celle rapportee pour I'enzyn~e du foie de rat, alors que la constante de fixation (IIK,) et la constante de la vitesse d'inactivation (kin,,,) sont toutes les deux semblables aux valeurs rapportkes pour l'enzyme cytoplasmique humain. La(RS)-abutyrolactone (5) inhibe aussi I'enzyme d'une faqon irrtversible, mais son efficacitt est beaucoup plus faible (IC,, de 2 nM). On a dktermink les valeurs de K, (9 mM) et de kin,,, (0,0078 s-I) du composk 5. Les rtsultats montrent que les valeurs de kin,,( des composts 1 et 5 different par un facteur de 2,5 alors que la valeur de K, du compost 5 est 1.8 x lo5 fois plus tlevke que celle du compost 1. On montre donc que le noyau p-lactone est la seule caracttristique structurale essentielle du composk 1 pour I'inactivation irrkversible de la synthase HMG-CoA; toutefois, par comparaison avec le compost 5, les autres fonctions augmentent la fixation du compost 1 B I'enzyme.

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Human Cytoplasmic 3-Hydroxy-3-methylglutaryl Coenzyme A Synthase: Expression, Purification, and Characterization of Recombinant Wild-Type and Cys129 Mutant Enzymes

Cited by 51 publications

References 0 publications

Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

3-Hydroxy-3-methylglutaryl-CoA Synthase

Irreversible inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from yeast by F-244 and (RS)-β-butyrolactone

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