Substrate Stereochemistry of 3‐Hydroxy‐3‐Methylglutaryl‐Coenzyme A Synthase

Abstract

Synthetic specimens of R‐[2H1, 3H1]acetate and of S‐[2H1, 3H1]acetate were converted into coenzyme A thiolesters. Each of these thiolesters was condensed with acetoacetyl‐CoA on 3‐hydroxy‐3‐methyl‐glutaryl‐CoA synthase. The products, which were found to be hydroxymethylglutaryl‐phosphopantetheines owing to a secondary cleavage of the coenzyme A moieties, were reduced chemically to 3R mevalonates. Each mevalonate specimen, after mixing with [2‐14C]mevalonate, was converted into squalene and cholesterol by a rat liver preparation. Each cholesterol specimen was converted into androst‐1,4‐diene‐3,17‐dione by incubation with a strain of Mycobacterium phlei in the presence of an inhibitor. When the procedures 4 and 5 were carried out with authentic specimens of 2R‐ and 2S‐[2‐3H1]mevalonate, it was found that the androstadienedione had lost nearly all tritium derived from the 2S‐mevalonate but had retained nearly all tritium (relative to 14C) derived from the 2R‐mevalonate. The androstadienedione derived, by procedures 1–5, from R‐acetate showed a loss of more than half of its tritium (relative to 14C). The androstadienedione derived by the same procedure from S‐acetate lost less than half of its tritium. It is concluded that the reaction on hydroxymethylglutaryl‐CoA synthase is stereospecific and is associated with an intramolecular hydrogen isotope effect. If this effect is normal (kH/kD>1) then the condensation proceeds with inversion of configuration at the methyl group.