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

Daniel J Darley, D S Butler, S J Prideaux, T W Thornton, A D Wilson, Timothy J Woodman, Michael D Threadgill, Matthew D Lloyd

Research output: Contribution to journalArticle

24 Citations (Scopus)

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 H-2-labelled substrate showed that loss of the alpha-proton was a prerequisite for chiral inversion. Reactions conducted in (H2O)-H-2 indicated that reprotonation was not stereospecific. These results are the first mechanistic study on any recombinant mammalian alpha-methylacyl-CoA racemase.
Original languageEnglish
Pages (from-to)543-552
Number of pages10
JournalOrganic and Biomolecular Chemistry
Volume7
Issue number3
Early online date28 Nov 2008
DOIs
Publication statusPublished - 7 Feb 2009

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Isotopes
enzymes
isotopes
Substrates
Enzymes
synthesis
inversions
Equilibrium constants
metabolism
Coenzyme A
Metabolism
Escherichia coli
lipids
Protons
Prostatic Neoplasms
drugs
cancer
proteins
Lipids
protons

Cite this

Synthesis and use of isotope-labelled substrates for a mechanistic study on human α-methylacyl-CoA racemase 1A (AMACR; P504S). / Darley, Daniel J; Butler, D S; Prideaux, S J; Thornton, T W; Wilson, A D; Woodman, Timothy J; Threadgill, Michael D; Lloyd, Matthew D.

In: Organic and Biomolecular Chemistry, Vol. 7, No. 3, 07.02.2009, p. 543-552.

Research output: Contribution to journalArticle

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