Identification of a critical cysteine in EcoRI DNA methyltransferase by mass spectrometry.
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.
Everett
E A
EA
Department of Chemistry, University of California, Santa Barbara 93106.
Falick
A M
AM
Reich
N O
NO
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004262:DNA Restriction Enzymes / Q000235:genetics; D015246:Deoxyribonuclease EcoRI / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000378:metabolism
D004151:Dipeptides; D010446:Peptide Fragments; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D003545:Cysteine; D005033:Ethylmaleimide