Cytochrome c specific methylase from wheat germ.
The cytochromes c of plants (e.g., wheat germ) possess two trimethyllysines, residues 72 and 86. In order to investigate the nature of these methylations, we have purified a cytochrome c specific methylase S-adenosylmethionine: protein(lysine) N-methyltransferase (protein methylase III) from wheat germ 135-fold. The in vitro site of methylation by both the purified enzyme and crude wheat germ extract toward various forms of horse heart cytochrome c was localized by two dimensional peptide mapping, Aminex A-5 column peptide analysis, and CNBr cleavage analysis to be the residue 72 lysine. However, no additional sites, in particular residue 86, were seen to be methylated. Although the enzyme is highly specific toward cytochrome c as an in vitro protein substrate, avian cytochromes c are seen to be much better substrates than those from mammalian sources. The enzyme possesses an extremely low Km for apocytochrome c (1.21 microM), suggesting that methylation may occur before heme attachment in vivo. Various S-adenosyl-L-homocysteine analogues were tested for their inhibitor capability toward the enzyme; it was observed that only the D and L forms of S-adenosylhomocysteine are inhibitors while analogues modified in the adenine or homocysteine moieties do not possess inhibitory capability. Results from the Aminex A-5 column chromatography of horse heart cytochrome c chymotryptic digest showed the N epsilon-methyl-, N epsilon-dimethyl-m and N epsilon-trimethyllysine forms of the residue 68-74 peptide to elute earlier than the unmethylated form. This results suggest that the methylated peptides are less basic than the unmethylated form.
D003574:Cytochrome c Group / Q000378:metabolism*; D011495:Histone-Lysine N-Methyltransferase / Q000302:isolation & purification*; D008239:Lysine / Q000378:metabolism; D011496:Protein Methyltransferases / Q000302:isolation & purification*; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D014908:Triticum / Q000201:enzymology*
D003574:Cytochrome c Group; D012435:S-Adenosylhomocysteine; D011496:Protein Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine