Analysis of enzyme reactions using NMR techniques: A case study with α-methylacyl-CoA racemase (AMACR)

Tim Woodman; Matthew Lloyd

doi:10.1016/bs.mie.2023.07.005

Analysis of enzyme reactions using NMR techniques: A case study with α-methylacyl-CoA racemase (AMACR)

Tim Woodman, Matthew Lloyd

Research output: Chapter or section in a book/report/conference proceeding › Chapter or section

2 Citations (SciVal)

Abstract

α-Methylacyl-CoA racemase (AMACR; P504S) catalyzes the conversion of R-2-methylacyl-CoA esters into their corresponding S-2-methylacyl-CoA epimers enabling their degradation by β-oxidation. The enzyme also catalyzes the key epimerization reaction in the pharmacological activation pathway of ibuprofen and related drugs. AMACR protein levels and enzymatic activity are increased in prostate cancer, and the enzyme is a recognized drug target. Key to the development of novel treatments based on AMACR inhibition is the development of functional assays. Synthesis of substrates and purification of recombinant human AMACR are described. Incubation of R- or S-2-methylacyl-CoA esters with AMACR in vitro resulted in formation of epimers (at a near 1–1 ratio at equilibrium) via removal of their α-protons to form an enolate intermediate followed by reprotonation. Conversion can be conveniently followed by incubation in buffer containing ²H ₂O followed by ¹H NMR analysis to monitor conversion of the α-methyl doublet to a single peak upon deuterium incorporation. Incubation of 2-methylacyl-CoA esters containing leaving groups results in an elimination reaction, which was also characterized by ¹H NMR. The synthesis of substrates, including a double labeled substrate for mechanistic studies, and subsequent analysis is also described.

Original language	English
Title of host publication	Modern Methods of Drug Design and Development
Editors	Matthew Lloyd
Place of Publication	New York, U. S. A.
Publisher	Elsevier Academic Press Inc
Chapter	6
Pages	159-209
Number of pages	51
ISBN (Electronic)	9780443158728
ISBN (Print)	9780443158711
DOIs	https://doi.org/10.1016/bs.mie.2023.07.005
Publication status	Published - 19 Oct 2023

Publication series

Name	Methods in Enzymology
Volume	690
ISSN (Print)	0076-6879
ISSN (Electronic)	1557-7988

Bibliographical note

Funding Information:
This work was supported at various times by EU grant QLG3-CT-2002–00696 (Refsum's disease: Diagnosis, Pathology and Treatment), Cancer Research UK, Prostate Cancer UK (grants S10–03 and PG14–009) and Biochemical Society Biochemical Summer Vacation Studentships. The authors gratefully acknowledge the Material and Chemical Characterisation Facility (MC2) at the University of Bath (doi.org/10.15125/mx6j-3r54) for technical support and assistance in this work. Parts of this work have been reproduced with permission from Royal Society of Chemistry publications. We thank Constant Systems, Daventry UK for providing the photo of the “one Shot” shown in Fig. 1B. The authors are part of the Cancer Research @ Bath (CR@B) network.

Funding Information:
This work was supported at various times by EU grant QLG3-CT-2002–00696 (Refsum’s disease: Diagnosis, Pathology and Treatment), Cancer Research UK, Prostate Cancer UK (grants S10–03 and PG14–009) and Biochemical Society Biochemical Summer Vacation Studentships. The authors gratefully acknowledge the Material and Chemical Characterisation Facility (MC 2 ) at the University of Bath (doi.org/10.15125/mx6j-3r54) for technical support and assistance in this work. Parts of this work have been reproduced with permission from Royal Society of Chemistry publications. We thank Constant Systems, Daventry UK for providing the photo of the “one Shot” shown in Fig. 1B . The authors are part of the Cancer Research @ Bath (CR@B) network.

Publisher Copyright:
© 2023

Keywords

Acyl-CoA esters
Chemical synthesis
Colorimetric substrate
Fluorine
Ibuprofen
NMR assays
α-Methylacyl-CoA racemase (AMACR, P504S)

ASJC Scopus subject areas

Molecular Biology
Biochemistry

UN SDGs

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

Access to Document

10.1016/bs.mie.2023.07.005

Cite this

Analysis of enzyme reactions using NMR techniques: A case study with α-methylacyl-CoA racemase (AMACR). / Woodman, Tim ; Lloyd, Matthew.
Modern Methods of Drug Design and Development. ed. / Matthew Lloyd. New York, U. S. A.: Elsevier Academic Press Inc, 2023. p. 159-209 (Methods in Enzymology; Vol. 690).

Research output: Chapter or section in a book/report/conference proceeding › Chapter or section

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AB - α-Methylacyl-CoA racemase (AMACR; P504S) catalyzes the conversion of R-2-methylacyl-CoA esters into their corresponding S-2-methylacyl-CoA epimers enabling their degradation by β-oxidation. The enzyme also catalyzes the key epimerization reaction in the pharmacological activation pathway of ibuprofen and related drugs. AMACR protein levels and enzymatic activity are increased in prostate cancer, and the enzyme is a recognized drug target. Key to the development of novel treatments based on AMACR inhibition is the development of functional assays. Synthesis of substrates and purification of recombinant human AMACR are described. Incubation of R- or S-2-methylacyl-CoA esters with AMACR in vitro resulted in formation of epimers (at a near 1–1 ratio at equilibrium) via removal of their α-protons to form an enolate intermediate followed by reprotonation. Conversion can be conveniently followed by incubation in buffer containing 2H 2O followed by 1H NMR analysis to monitor conversion of the α-methyl doublet to a single peak upon deuterium incorporation. Incubation of 2-methylacyl-CoA esters containing leaving groups results in an elimination reaction, which was also characterized by 1H NMR. The synthesis of substrates, including a double labeled substrate for mechanistic studies, and subsequent analysis is also described.

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Analysis of enzyme reactions using NMR techniques: A case study with α-methylacyl-CoA racemase (AMACR)

Abstract

Publication series

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

Other files and links

Fingerprint

Avance III 400 MHz Nuclear Magnetic Resonance (NMR) Spectrometer (9West)

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

MC2-Mass Spectrometry (MS)

Cite this

Analysis of enzyme reactions using NMR techniques: A case study with α-methylacyl-CoA racemase (AMACR)

Abstract

Publication series

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

Other files and links

Fingerprint

Equipment

Avance III 400 MHz Nuclear Magnetic Resonance (NMR) Spectrometer (9West)

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

MC2-Mass Spectrometry (MS)

Cite this