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

Abstract

Kinetic properties of protein methylase I1 (S-adenosy1methionine:protein-carboxyl methyltransferase, 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-Lhomocysteine (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-carboxymethy1thio)butyric acid exerted a significant inhibition.AdoHcy, the demethylated product of AdoMet, exerts a competitive inhibition on the reaction (Ki = 0.65 pM); 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 a-carbon in the binding to the enzyme protein. 5'-Methylthioadenosine, a metabolic product of AdoMet, inhibited competitively the reaction (Ki = 41 pM) while 5'-methylthioadenosine derivatives, such as n-butylthioadenosine, isobutylthioadenosine and thioethanoladenosine, failed to exert any inhibition.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 (Glul,16, Tyrl)] ( M , = 22600) exerts a relevant non-competitive inhibition, while none of the tested synthetic polypeptides is active as substrate.Protein methylase I1 (S-adenosylmethionine : protein-carboxyl methyltransferase) catalyzes the transfer of the methyl group from S-adenosylmethionine (AdoMet) to free carboxyl groups of aspartyl and/or glutamyl residues in protein, yielding carboxyl methyl esters [l -31. In turn protein methyl esters undergo rapid and spontaneous hydrolysis into methanol at physiological pH and temperature [4,5]. The enzyme is widely distributed in nature and has been shown to be present in the cytosolic fraction of most mammalian tissues [6,7].

Effect of 5'‐methylthioadenosine on in vivo methyl esterification of human erythrocyte membrane proteins

“…5'-Isobutylthioadenosine, a synthetic analog of MTA endowed with antiproliferative action [ 181 which does not inhibit PM II in vitro [20], exerted a significant inhibition on the reaction in the system investigated. The in vivo effect of this drug could be due to the action of the adenine product of phosphorolytic cleavage of SIBA [30].…”

“…The inhibition exerted in vitro by MTA on PM II is of particular interest [20,21]. To investigate the control mechanism operated by MTA on the enzytnatic methyl esterification of membrane proteins in the living cells, the effect of the compound on in vivo methyl esterification of erythrocyte membrane proteins has been analyzed.…”

Enzymatic methyl esterification of erythrocyte membrane proteins is impaired in chronic renal failure. Evidence for high levels of the natural inhibitor S-adenosylhomocysteine.

“…AdoHcy is the natural by-product of the methyl transfer reaction from AdoMet to methyl accepting proteins, and also a potent inhibitor of AdoMet-dependent reactions (26)(27)(28)(29).…”

“…Type II MTase utilizes S-adenosylmethionine (AdoMet), as the methyl donor (25). The reaction generates S-adenosylhomocysteine (AdoHcy), which represents the natural inhibitor of this methyl transfer reaction (26,27), as well as of most AdoMet-dependent methylations (28,29 PMSF, and centrifuged at 10,000 g for 30 min. The membrane pellet was subsequently washed twice with the same ipotonic solution at pH 7.2 and 6.8, respectively.…”

Repair of O6-ethylguanine in DNA by a chromatin fraction from rat liver: transfer of the ethyl group to an acceptor protein.

“…Because the w-carboxyl groups of aspartate and glutamate can also accept alkyl groups (22), authentic markers ofethyl glutamate and ethyl aspartate were also used as chromatographic markers.…”