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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.