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

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Abstract
Kinetic properties of [calf thymus] protein methylase II (S-adenosylmethionine: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 RNase, a model substrate for the enzyme, was subjected to specific cleavage by CNBr, trypsin and performic acid oxidation. Several polypeptide fragments derived were then separated by molecular sieve chromatography on a Sephadex G-25 column. The method was very simple and gave good yields. Km values for these polypeptides and other protein substrates were determined. While Km values for the isolated peptides range generally between 4.8 and 0.7 .times. 10-3 M, those of native RNase, luteinizing hormone and follicle-stimulating hormone were determined to be 4.0 .times. 10-4, 5.0 .times. 10-5 and 0.77 .times. 10-5 M, respectively. Sites of enzymatic methylation of the native RNase were also investigated. Although polypeptides derived from the C-terminal and N-terminal regions of the molecule accepted methyl groups, they were unable to undergo enzymatic methylation when native molecule was used as the substrate, indicating that within the native RNase these regions are in a conformation which do not allow them to be methylated by protein methylase II under the present assay conditions.