Mechanism and Structural Requirements for Transformation of Substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol Methyl Transferase From Saccharomyces Cerevisiae

Biochim Biophys Acta. 1996 Feb 16;1299(3):313-24. doi: 10.1016/0005-2760(95)00218-9.

Abstract

The mechanism of action and active site of the enzyme (S)-adenosyl-L-methionine:delta 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, 14 alpha-methyl or 9 beta,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, kappa H/kappa 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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Fractionation
  • Chromatography, High Pressure Liquid
  • Enzyme Inhibitors
  • Kinetics
  • Methylation
  • Methyltransferases / antagonists & inhibitors
  • Methyltransferases / metabolism*
  • Microsomes / enzymology
  • Models, Biological
  • S-Adenosylmethionine / metabolism*
  • Saccharomyces cerevisiae / enzymology*
  • Solubility
  • Sterols / metabolism*
  • Substrate Specificity

Substances

  • Enzyme Inhibitors
  • Sterols
  • S-Adenosylmethionine
  • Methyltransferases
  • delta 24-sterol methyltransferase