A fluorescence-based assay for N-myristoyltransferase activity

Anal Biochem. 2012 Feb 1;421(1):342-4. doi: 10.1016/j.ab.2011.10.013. Epub 2011 Oct 14.

Abstract

N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Acyltransferases / analysis*
  • Acyltransferases / antagonists & inhibitors
  • Acyltransferases / genetics
  • Acyltransferases / metabolism
  • Coenzyme A
  • Coumarins
  • Fluorescence
  • Fluorescent Dyes
  • Humans
  • Kinetics
  • Myristic Acids / metabolism
  • Protein Processing, Post-Translational

Substances

  • Coumarins
  • Fluorescent Dyes
  • Myristic Acids
  • N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide
  • Acyltransferases
  • glycylpeptide N-tetradecanoyltransferase
  • Coenzyme A