The role of cysteine-150 in the structure and activity of rat liver S-adenosyl-L-methionine synthetase.
The present paper reports the tryptic digestion of N-ethylmaleimide-treated S-adenosyl-L-methionine synthetase (high- and low-Mr forms) and the isolation of the modified peptides by h.p.l.c. There is only one site modified after 5 min incubation, and the modification at this site correlates with the main activity decrease. The amino acid composition of this peptide was determined, and its localization in the sequence shows the modified residue as cysteine-150, which is located close to the putative ATP-binding site. Modification of the enzyme for 20 min led to the appearance of a second labelled peptide, which seems to be responsible for about a further 10% of the activity loss. The modification by N-ethylmaleimide of the enzyme was partially prevented in the presence of adenosine 5'-[beta gamma-imido]triphosphate and methionine, further supporting the hypothesis that the modified residues lie within the active site. Urea treatment of the enzyme, followed by modification with N-ethylmaleimide, produces the modification of 7 of the 10 cysteine residues present in the sequence. The results obtained were the same for either of the isoforms.
Pajares
M A
MA
Instituto de Investigaciones Biomédicas del Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Corrales
F J
FJ
Ochoa
P
P
Mato
J M
JM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D005033:Ethylmaleimide / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008716:Methionine Adenosyltransferase / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011485:Protein Binding; D051381:Rats; D014357:Trypsin; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D010446:Peptide Fragments; D014508:Urea; D008716:Methionine Adenosyltransferase; D014357:Trypsin; D003545:Cysteine; D005033:Ethylmaleimide