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Anal Biochem. 2014 Apr 1;450:11-9. doi: 10.1016/j.ab.2013.12.026. Epub 2013 Dec 27.

A sensitive mass spectrum assay to characterize engineered methionine adenosyltransferases with S-alkyl methionine analogues as substrates.

Author information

1
Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA; Program of Pharmacology, Weill Graduate School of Medical Science, Cornell University, New York, NY 10021, USA.
2
Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
3
Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Electronic address: luom@mskcc.org.

Abstract

Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-l-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAMs) into the SAM analogues with engineered MATs. Here we describe a general sensitive activity assay for engineered MATs by converting the reaction products into S-alkylthioadenosines, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification. With this assay, 40 human MAT mutants were evaluated against 7 SAAMs as potential substrates. The structure-activity relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower Km value in contrast to native MATs), the gained activity toward the bulky SAAMs stems from their ability to maintain the desired linear SN2 transition state (reflected by higher kcat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAMs.

KEYWORDS:

Epigenetics; LC–MS/MS; MAT; Methyltransferase; S-Adenosyl-l-methionine

PMID:
24374249
PMCID:
PMC3947680
DOI:
10.1016/j.ab.2013.12.026
[Indexed for MEDLINE]
Free PMC Article

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