A comparison of kinetic parameters of polypeptide substrates for protein methylase II.

Kinetic properties of protein methylase II (S-adenosymethionine:protein O-methyltransferase, EC 2.1.1.24) which methylates (esterifies) the free carboxyl side chains of amino acids in proteins was studied using various polypeptides as methyl acceptor substrates. Bovine pancreatic ribonuclease, a model substrate for the enzyme, was subjected to specific cleavage by cyanogen bromide, trypsin, and performic acid oxidation. Several polypeptide fragments derived were then separated by molecular sieve chromatography on a column of Sephadex G-25. The method was found to be very simple and gave good yields. Km values for these polypeptides as well as a few other protein substrates were determined. While Km values for the isolated peptides range generally between 4.8 and 0.7 X 10-3 M, those of native bovine panreatic ribonuclease, luteinizing hormone, and follicle-stimulating hormone were determined to be 4.0 X 10-4, 5.0 X 10-5, and 0.77 X 10-5, respectively. Sites of enzymatic methylation of the native ribonuclease were also investigated. Although polypeptides derived from the C-terminal and N-terminal regions of the molecule were found to accept methyl groups, they were unable to under go enzymatic methylation when native molecule was used as the substrate indicating that within the native ribonuclease these regions are in a conformation which do not allow them to be methylated by protein methylase II under the present assay conditions.