Purification and characterization of enzymes from Euglena gracilis that methylate methionine and arginine residues of cytochrome c.
Two forms of cytochrome c-specific methyltransferases from Euglena gracilis were purified approximately 100- and 50-fold, respectively, using DEAE-cellulose and gel-filtration chromatography. The methylation product of enzyme I was identified as S-methylmethionine and that of enzyme II as NG-monomethylarginine. Both enzymes were located in the cytosol and exhibit maximum activity at pH 7.0. Among the various proteins tested as substrates, the enzymes were highly specific toward cytochrome c. Various types of histones, in particular, were not modified by either enzyme. The molecular weights of enzyme I and II were 28,000 and 36,000, respectively. Various S-adenosyl-L-homocysteine analogs were tested for their inhibitory activity toward the enzymes. Only the D- and L-isomers of S-adenosylhomocysteine and sinefungin were significantly inhibitory. The Ki values for S-adenosyl-L-homocysteine were 8.13 X 10(-6) and 1.17 X 10(-5) M for enzyme I and II, respectively. Two-dimensional peptide mapping revealed the methylation site of enzyme I to be the methionine residue at position 65 while that of enzyme II to be the arginine residue at position 38. The methylation of either methionine or arginine residues by enzyme I and II, respectively, lowers the isoelectric point (pI) of cytochrome c: 9.23, 9.33, and 10.06 for S-methylmethionine-, NG-monomethylarginine-modified, and unmodified cytochrome c, respectively.
Farooqui
J Z
JZ
Tuck
M
M
Paik
W K
WK
D002918:Chymotrypsin; D003574:Cytochrome c Group / Q000378:metabolism*; D005056:Euglena gracilis / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011496:Protein Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D011484:Protein-Arginine N-Methyltransferases / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D003574:Cytochrome c Group; D010446:Peptide Fragments; D011496:Protein Methyltransferases; C044109:cytochrome C-methionine methyltransferase; D011484:Protein-Arginine N-Methyltransferases; D002918:Chymotrypsin