Purification and Inhibition of Hydroxymethylglutaryl Coenzyme A Synthase

Abstract

Hydroxymethylglutaryl-CoA synthase (HMG-CoA synthase) catalyzes the formation of HMG-CoA from acetyl-CoA and acetoacetyl-CoA. F-244 (1), a naturally occurring J3-lactone isolated from Fusarium sp. ATCC 20788 and other species, is known to be a potent and specific inhibitor ofHMG-CoA synthase isolated from rat liver. 1Ji,, HO,.,._.. .. , '• .. ,....13_.,., ;=( 0 14 0 0 (1) (2) This thesis describes the 48 fold purification of HMG-CoA synthase from bakers yeast in a three step procedure involving ethanol fractionation followed by ammonium sulfate precipitation and then hydroxylapatite chromatography. This procedure was found to be reproducible and yields a preparation of specific activity 0.14 units (j...tmolfmin)/mg in an overall yield of 8%. In our study, F-244 was found to be a potent irreversible inhibitor of HMGCoA synthase isolated from bakers yeast, with an IC50 value of 0.009 j...tM. This value is almost identical to the inhibitory activity of F-244 on rat liver HMG-CoA synthase that has been reported in the literature. Tritium labeled F-244 was prepared, for the first time, by feeding methyl-[3H]methionine to cultures of Fusarium sp. The [15, 16, 17, 18-3H] F-244 isolated had a specific activity of 1.3 x 106 DPM/mg. This tritiated F-244 was then used as an affinity.label for HMG-CoA synthase. Attempts to isolate the enzyme-inhibitor complex were unsuccessful due to the low level of radioactivity associated with the tritiated F-244. HMG-CoA synthase was also shown to be inhibited in a time-dependent irreversible manner by {±)-(3-butyrolactone (2). The rate of inactivation (~) was found to be 0.4697 s-1 and the inhibition constant (K1) was found to be 9 mM. The inactivation was found to be irreversible over several hours.