Synthesis and use of isotope-labelled substrates for a mechanistic study on human alpha-methylacyl-CoA racemase 1A (AMACR; P504S)

Org Biomol Chem. 2009 Feb 7;7(3):543-52. doi: 10.1039/b815396e. Epub 2008 Nov 28.

Abstract

Alpha-Methylacyl-CoA racemase (AMACR) is an important enzyme for the metabolism of branched-chain lipids and drugs. The enzyme is over-expressed in prostate and other cancers. AMACR 1A, the major splice variant, was purified from recombinant E. coli cells as a His-tag protein. Purified enzyme catalysed chiral inversion of both S- and R-2-methyldecanoyl-CoA, with an equilibrium constant of 1.09 +/- 0.14 (2S/2R). Reactions with (2)H-labelled substrate showed that loss of the alpha-proton was a prerequisite for chiral inversion. Reactions conducted in (2)H(2)O indicated that reprotonation was not stereospecific. These results are the first mechanistic study on any recombinant mammalian alpha-methylacyl-CoA racemase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl Coenzyme A / chemical synthesis*
  • Acyl Coenzyme A / chemistry
  • Acyl Coenzyme A / metabolism*
  • Biocatalysis
  • Escherichia coli / genetics
  • Humans
  • Isoenzymes / metabolism
  • Isotope Labeling
  • Racemases and Epimerases / metabolism*
  • Recombinant Proteins / metabolism
  • Stereoisomerism

Substances

  • Acyl Coenzyme A
  • Isoenzymes
  • Recombinant Proteins
  • Racemases and Epimerases
  • alpha-methylacyl-CoA racemase