Mechanism and structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:Δ24(25)sterol methyl transferase from Saccharomyces cerevisiae

Mylavarapu Venkatramesh, De An Guo, Zhonghua Jia, W. David Nes

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The mechanism of action and active site of the enzyme (S)-adenosyl-L-methionine:Δ24(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 [3-3H]zymosterol with AdoMet or [methyl-2H3]AdoMet confirmed the operation of a random mechanism, kH/kD= 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.

Original languageEnglish
Pages (from-to)313-324
Number of pages12
JournalBiochimica et Biophysica Acta - Lipids and Lipid Metabolism
Issue number3
StatePublished - Jan 1 1996



  • (S. Cerevisiae)
  • Kinetics
  • Membrane fluidity
  • Sterol biomethylation

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