Identification of a critical cysteine in EcoRI DNA methyltransferase by mass spectrometry

J Biol Chem. 1990 Oct 15;265(29):17713-9.

Abstract

EcoRI DNA methyltransferase (MTase) is rapidly inactivated by N-ethylmaleimide with concomitant incorporation of 2 mol of N-ethyl[2-3H]maleimide/mol of functional monomer. Preincubation of the enzyme with either S-adenosylmethionine or DNA reduces the rate of activity loss, whereas preincubation with DNA and the S-adenosylmethionine analog sinefungin completely protects the enzyme from inactivation. An endo proteinase Glu-C digest of N-ethyl[2-3H]maleimide-modified enzyme was prepared and separated by high pressure liquid chromatography. Modified and unmodified cysteine-containing peptides were located and identified by radioactivity, mass spectrometry, and tandem mass spectrometry. In the absence of any ligands, cysteines 25, 116, and 223 are modified by N-ethylmaleimide; in the presence of DNA and sinefungin, Cys-223 is essentially unmodified. Thus, N-ethylmaleimide modification of Cys-223 in EcoRI DNA MTase is responsible for the loss of enzyme activity. Cys-223 is preceded by Asn, and this (or Cys-Asn) occurs with high frequency in adenine and cytosine (N-4) DNA MTases. Direct involvement of cysteine in methyl transfer reactions to adenine N-6 and cytosine N-4 is supported by the similarity of the reactions catalyzed by adenine N-6 and cytosine N-4 DNA MTases, the frequent presence of Asn-flanking Cys, and the importance of Cys-223 to EcoRI MTase function.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Chromatography, High Pressure Liquid
  • Cysteine*
  • DNA Restriction Enzymes / genetics
  • Deoxyribonuclease EcoRI / antagonists & inhibitors
  • Deoxyribonuclease EcoRI / genetics
  • Deoxyribonuclease EcoRI / metabolism*
  • Dipeptides / analysis
  • Escherichia coli / enzymology
  • Ethylmaleimide / pharmacology
  • Kinetics
  • Mass Spectrometry / methods
  • Molecular Sequence Data
  • Peptide Fragments / isolation & purification
  • Staphylococcus aureus / metabolism

Substances

  • Dipeptides
  • Peptide Fragments
  • DNA Restriction Enzymes
  • Deoxyribonuclease EcoRI
  • Cysteine
  • Ethylmaleimide