title: A visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase.
abstract: 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase catalyzes the first physiologically irreversible step in biosynthesis of isoprenoids and sterols from acetyl-CoA. Inhibition of enzyme activity by beta-lactone-containing natural products correlates with substantial diminution of sterol synthesis, identifying HMG-CoA synthase as a potential drug target and suggesting that identification of effective inhibitors would be valuable. A visible wavelength spectrophotometric assay for HMG-CoA synthase has been developed. The assay uses dithiobisnitrobenzoic acid (DTNB) to detect coenzyme A (CoASH) release on acetylation of enzyme by the substrate acetyl-CoA, which precedes condensation with acetoacetyl-CoA to form the HMG-CoA product. The assay method takes advantage of the stability of recombinant enzyme in the absence of a reducing agent. It can be scaled down to a 60 microl volume to allow the use of 384-well microplates, facilitating high-throughput screening of compound libraries. Enzyme activity measured in the microplate assay is comparable to values measured by using conventional scale spectrophotometric assays with the DTNB method (412 nm) for CoASH production or by monitoring the use of a second substrate, acetoacetyl-CoA (300 nm). The high-throughput assay method has been successfully used to screen a library of more than 100,000 drug-like compounds and has identified both reversible and irreversible inhibitors of the human enzyme.
authors.affiliation: Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA.
authors.forename: D
authors.initials: D
authors.lastname: Andrew Skaff
authors.forename: Henry M
authors.initials: HM
authors.lastname: Miziorko
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008027:Light; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D013053:Spectrophotometry / Q000379:methods*; D013379:Substrate Specificity / Q000187:drug effects; D013729:Terpenes / Q000378:metabolism; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D013729:Terpenes; D000105:Acetyl Coenzyme A; D004228:Dithionitrobenzoic Acid; D006904:Hydroxymethylglutaryl-CoA Synthase