MECHANISM AND STRUCTURAL REQUIREMENTS FOR TRANSFORMATION OF SUBSTRATES BY THE (S)-ADENOSYL-L-METHIONINE : DELTA 24(25)-STEROL METHYL TRANSFERASE FROM SACCHAROMYCES CEREVISIAE

Abstract The mechanism of action and active site of the enzyme (S)- adenosyl- l -methionine :Δ 24(25) - sterol methyl transferase (SMT) from Saccharomyces cerevisiae strain GL7 have been probed with AdoMet, (S)- adenosyl- l -homocysteine , a series of 35 sterol substrates as acceptor molecules and a series of 10 substrate and high energy intermediate (HEI) sterol analogues as inhibitors of biomethylation. The SMT was found to be selective for sterol, both regio- and stereochemically. The presence of an unhindered 24,25-bond, an equatorially-oriented polar group at C-3 (which must act as a proton acceptor) attached to a planar nucleus and a freely rotating side chain were obligatory structural features for sterol binding/catalysis; no essential requirement or significant harmful effects could be found for the introduction of an 8(9)-bond, 14α-methyl or 9β,19-cyclopropyl group. Alternatively, methyl groups at C-4 prevented productive sterol binding to the SMT. Initial velocity, product inhibition, and dead-end experiments demonstrated a rapid-equilibrium random bi bi mechanism. Deuterium isotope effects developed from SMT assays containing mixtures of [3- 3 H]zymosterol with AdoMet or [ methyl - 2 H 3 ]AdoMet confirmed the operation of a random mechanism, k H k D = 1.3 . From this combination of results, the spatial relationship of the sterol substrate to AdoMet could be approximated and the topology of the sterol binding to the SMT thereby formulated.