title: Synthesis and use of isotope-labelled substrates for a mechanistic study on human alpha-methylacyl-CoA racemase 1A (AMACR; P504S).
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.
authors.affiliation: Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Darley
authors.forename: Danica S
authors.initials: DS
authors.lastname: Butler
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Prideaux
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Thornton
authors.forename: Abigail D
authors.initials: AD
authors.lastname: Wilson
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Woodman
authors.forename: Michael D
authors.initials: MD
authors.lastname: Threadgill
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Lloyd
mesh_terms: D000214:Acyl Coenzyme A / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007553:Isotope Labeling; D019998:Racemases and Epimerases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D000214:Acyl Coenzyme A; D007527:Isoenzymes; D011994:Recombinant Proteins; D019998:Racemases and Epimerases; C108352:alpha-methylacyl-CoA racemase