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

doi:10.1039/b815396e

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

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

33 Citations (SciVal)

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 language	English
Pages (from-to)	543-552
Number of pages	10
Journal	Organic and Biomolecular Chemistry
Volume	7
Issue number	3
Early online date	28 Nov 2008
DOIs	https://doi.org/10.1039/b815396e
Publication status	Published - 7 Feb 2009

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Access to Document

10.1039/b815396e

Cite this

@article{ec907d6935494c8aa3493bae2805d114,

title = "Synthesis and use of isotope-labelled substrates for a mechanistic study on human α-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 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.",

author = "Darley, \{Daniel J\} and Butler, \{D S\} and Prideaux, \{S J\} and Thornton, \{T W\} and Wilson, \{A D\} and Woodman, \{Timothy J\} and Threadgill, \{Michael D\} and Lloyd, \{Matthew D\}",

year = "2009",

month = feb,

day = "7",

doi = "10.1039/b815396e",

language = "English",

volume = "7",

pages = "543--552",

journal = "Organic and Biomolecular Chemistry",

issn = "1477-0520",

publisher = "Royal Society of Chemistry",

number = "3",

}

TY - JOUR

T1 - Synthesis and use of isotope-labelled substrates for a mechanistic study on human α-methylacyl-CoA racemase 1A (AMACR; P504S)

AU - Darley, Daniel J

AU - Butler, D S

AU - Prideaux, S J

AU - Thornton, T W

AU - Wilson, A D

AU - Woodman, Timothy J

AU - Threadgill, Michael D

AU - Lloyd, Matthew D

PY - 2009/2/7

Y1 - 2009/2/7

N2 - 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.

AB - 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.

UR - https://www.scopus.com/pages/publications/58849124628

UR - http://dx.doi.org/10.1039/b815396e

U2 - 10.1039/b815396e

DO - 10.1039/b815396e

M3 - Article

SN - 1477-0520

VL - 7

SP - 543

EP - 552

JO - Organic and Biomolecular Chemistry

JF - Organic and Biomolecular Chemistry

IS - 3

ER -

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

Abstract

UN SDGs

Access to Document

Other files and links

Fingerprint

Isoforms as a Novel Target for Prostatic Cancer

Avance III 500 MHz Nuclear Magnetic Resonance (NMR) Spectrometer (9West)

MC2-Mass Spectrometry (MS)

MC2- Nuclear Magnetic Resonance (NMR)

Cite this

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

Abstract

UN SDGs

Access to Document

Other files and links

Fingerprint

Projects

Isoforms as a Novel Target for Prostatic Cancer

Equipment

Avance III 500 MHz Nuclear Magnetic Resonance (NMR) Spectrometer (9West)

MC2-Mass Spectrometry (MS)

MC2- Nuclear Magnetic Resonance (NMR)

Cite this