The inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase from yeast was compared for both F-244 (1) and (RS)-β -butyrolactone (5). F-244 exhibits irreversible inhibition with an IC50of 8 nM, similar to that reported for the rat liver enzyme, while the binding constant (1/K1) and inactivation rate constant (kinact) similar to values reported for the human cytoplasmic enzyme. (RS)-β-Butyrolactone (5) also irreversibly inhibits the enzyme, but with much lower efficiency (IC502 mM). The values forK1(9 mM) andkinact(0.0078 s−1) for5were determined. The results show thatkinactfor 5 and1differ by a factor of only 2.5, whileK1for5is higher by a factor of 1.8 × 105. Hence, the β-lactone ring is shown to be the sole essential structural feature in1for irreversible inactivation of HMG-CoA synthase; however, the remaining functionality enhances the binding of1to the enzyme relative to5.Keywords: HMG-CoA synthase, F-244, butyrolactone, irreversible, inhibitor.