Article Figures & Data
Figures
- FIG. 1.
Intermediates and enzymes of the mevalonate pathway for IPP biosynthesis.
- FIG. 2.
SDS-PAGE of fractions obtained during purification of E. faecalis HMG-CoA synthase. Lane 1, prestained standards of the indicated molecular weight; lane 2, cell extract; lane 3, cytosol; lane 4, Ni-NTA fraction.
- FIG. 3.
Sedimentation velocity ultracentrifugation of E. faecalis HMG-CoA synthase. (A) Sedimentation boundaries measured by using Rayleigh interference optics plotted against radial position. The data shown are for 25-min intervals and represent one-fifth of the data used in the analysis. The jagged lines represent the observed fringes, and the smooth lines represent the calculated best-fit distribution calculated by using Sedfit 8.3 and the Lamm equation model (23). (B) Best-fit c(s) sedimentation coefficient distribution, allowing for systematic time-invariant noise. The uncorrected values for the sedimentation coefficients for the minor and major peaks are 3.6 and 5.3 S, respectively.
- FIG. 4.
Effect of temperature, MgCl2 concentration, and hydrogen ion concentration. (Top) Temperature. Assays were conducted at the indicated temperatures under otherwise standard conditions. The inset shows selected data shown as an Arrhenius plot. (Middle) MgCl2.Assays were conducted at the indicated concentrations of MgCl2 under otherwise standard conditions. (Bottom) Hydrogen ion concentration. Assays were conducted in 50 mM sodium acetate, 50 mM glycine, 50 mM Tris, and 50 mM 2-(N-morpholino)ethanesulfonic acid at the indicated pH under otherwise standard conditions.
- FIG. 5.
Proposed mechanism for catalysis of the HMG-CoA synthase reaction. Active site residues of the avian mitochondrial enzyme include Glu95, Cys129, and His264. During the acetylation step, Cys129 attacks the carbonyl group of acetyl-CoA, forming an acetyl-S-intermediate (17). After the addition and condensation of acetoacetyl-CoA, HMG-CoA is released by hydrolysis. Glu95 acts as the general acid in the condensation step (8), and His264 anchors binding of acetoacetyl-CoA (16).
- FIG. 6.
Coupled conversion of acetyl-CoA to mevalonate catalyzed by E. faecalis acetoacetyl-CoA thiolase/HMG-CoA reductase and HMG-CoA synthase. Shown is the effect of hydrogen ion concentration on the conversion of acetyl-CoA to acetoacetyl-CoA (○), of HMG-CoA to mevalonate (□), and of acetyl-CoA to mevalonate (•). All assays employed 50 mM sodium acetate, 50 mM glycine, 50 mM Tris, and 50 mM 2-(N-morpholino)ethanesulfonic acid at the indicated pH. Assays of acetoacetyl-CoA thiolase and HMG-CoA reductase activity were conducted essentially as previously described (11), but under the above conditions. For the conversion of acetyl-CoA to mevalonate, the additions were: 1 mM acetyl-CoA, 0.4 mM NADPH, 9 nM E. faecalis acetoacetyl-CoA thiolase/HMG-CoA reductase subunit, and 16 nM E. faecalis HMG-CoA synthase.
Tables
- TABLE 1.
Selected sequences and conserved residues of animal and bacterial HMG-CoA synthasesa
Sequence set and sourceb Sequence (residue position) Set 1 (95) Animal LEVGTETIIDKSKxVK Bacterial VIVATESxIDxxKAxx Set 2 (129) Animal DTTNACYGGT Bacterial ExxxACYxAT Set 3 (264) Animal MIFHxPxxK Bacterial xxFHxPxCK ↵ a Capital letters indicate conserved residues. Residues in boldface are conserved in both animal and bacterial HMG-CoA synthases. An “x” indicates a nonconserved residue. Glu95 (8), Cys129 (17), and His264 (16) have been identified as catalytic residues in the avian enzyme. For E. faecalis HMG-CoA synthase these residues are Glu79, Cys111, and His233.
↵ b Animal cytosolic HMG-CoA synthases aligned are from rats, hamsters, humans, and chickens. Animal mitochondrial HMG-CoA synthases aligned are from mouse, rat, human, and pig. Aligned bacterial HMG-CoA synthase sequences are from Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus aureus, and a Streptomyces sp.
- TABLE 2.
Kinetic constants for selected HMG-CoA synthases
Source (reference) Km for acetyl-CoA (μM) Kmapp for acetoacetyl-CoA (μM) Vmax (μmol/min/mg) E. faecalis 350 10 10 Avian liver cytosol (7) 270 1.2 4.4 Blattella germanica (6) 1,500 <0.5 66 - TABLE 3.
Parameters for the partial reactions that model the first and third stages of the overall reaction catalyzed by HMG-CoA synthase
Source (reference) Acetyl-CoA bound (mol/mol of monomer)a Acetyl-CoA covalently bound (mol/mol of monomer)b Hydrolysis of acetyl-CoA Vmax (eu/mg) Km (μM) E. faecalis 1.2 ± 0.2c 0.60 ± 0.02c 0.02 8.5 Avian liver cytosol (7, 8) 1.1 0.63 0.016 11 ↵ a To determine the stoichiometry of acetyl-CoA binding, 100 μg of HMG-CoA synthase and 0.5 mM or 1 mM [1-14C]acetyl-CoA (specific activity, 150 cpm/nmol) in 50 mM Tris (pH 9.75) were combined in a volume 70 μl and incubated at room temperature for 5 min. The entire reaction mixture was then applied to a MicroSpin BioP-30 gel filtration column and centrifuged for 4 min at 1,000 × g. The filtrate was then assayed for protein concentration (5) and radioactivity. The stoichiometry was determined by dividing the moles of acetyl-CoA by the moles HMG-CoA synthase monomer present.
↵ b The stoichiometry of covalently bound acetyl-CoA is presented. HMG-CoA synthase (100 μg), 200 μg of bovine serum albumin as a carrier protein, and 0.5, 1.0, 2.0, or 3.0 mM [1-14C]acetyl-CoA (specific activity, 150 cpm/nmol) in 50 mM Tris buffer (pH 9.75) at a final volume of 150 μl were mixed on ice. After 2 min, 1 ml of 10% trichloroacetic acid was added, and each incubation mixture was applied to a glass fiber filter. Filters were washed with 10% trichloroacetic acid and then with 1 ml of ice-cold absolute ethanol, allowed to dry, and counted. The stoichiometry of covalently bound acetyl-CoA was determined by dividing the moles of acetyl-CoA by the moles HMG-CoA synthase monomer present.
↵ c Mean ± the standard deviation.
