Studies on Substrate Specificity of S-Adenosylmethionine: Protein-Carboxyl Methyltransferase from Calf Brain

Kinetic properties of protein methylase II (S-adenosylmethionine:protein-carboxyl methyltrans- ferase, EC 2.1.1.24), which methylates the free carboxyl groups of protein substrates, were studied with various analogs and derivatives of S-adenosyl-l-methionine (AdoMet) and S-adenosyl-l- homocysteine (AdoHcy), using corticotropin as methyl-accepting polypeptide. Experiments with methyl-labelled structural analogs of AdoMet showed that the replacement of the amino groups of AdoMet by hydroxyl groups, as well as the removal of the carboxyl group, results in a loss of methylating ability of the molecule. Deaminated and decarboxylated derivatives were also inactive as inhibitors. Among the sulfonium analogs tested, only S-adenosyl-l- (2-amino-4-carboxymethylthio)butyric acid exerted a significant inhibition. AdoHcy, the demethylated product of AdoMet, exerts a competitive inhibition on the reaction (Ki= 0.65 μM); the probable regulatory role of this inhibition will be discussed. The removal of the adenine amino group of the thioether resulted in a loss of the inhibitory effect. Experiments with the D-isomer of AdoHcy showed the relevance of the steric configuration of the α-carbon in the binding to the enzyme protein. 5′-Methylthioadenosine, a metabolic product of AdoMet, in- hibited competitively the reaction (Ki= 41 μM) while 5′-methylthioadenosine derivatives, such as n-butylthioadenosine, isobutylthioadenosine and thioethanoladenosine, failed to exert any in- hibition. Three synthetic polypeptides differing in amino acid composition and in chain length have also been tested as methyl-acceptor substrates and as inhibitors for the reaction. Only the copolymer of l-glutamic acid and l-tyrosine [poly(Glul1.16, Tyrl)] (Mr= 22600) exerts a relevant non-com- petitive inhibition, while none of the tested synthetic polypeptides is active as substrate.