Mechanistic studies of the processing of human S-adenosylmethionine decarboxylase proenzyme. Isolation of an ester intermediate.
Human S-adenosylmethionine decarboxylase is synthesized as a proenzyme that undergoes an autocatalytic cleavage reaction generating the alpha and beta subunits and forming the pyruvate prosthetic group, which is derived from an internal Ser residue (Ser-68). The mechanism of this processing reaction was studied using site-directed mutagenesis of conserved residues (His-243 and Ser-229) located close to the cleavage site. Mutant S229A failed to process, and mutant S229C cleaved very slowly, whereas mutant S229T processed normally, suggesting that the hydroxyl group of residue 229 is required for the processing reaction where Ser-229 may act as a proton acceptor. Mutant His-243A cleaved very slowly, forming a small amount of the correctly processed pyruvoyl enzyme but a much larger proportion of the alpha subunit with an amino-terminal Ser. The cleavage to form the latter was greatly enhanced by hydroxylamine. This result suggests that the N-O acyl shift needed for ester formation occurs normally in this mutant but that the next step, which is a beta-elimination reaction leading to the two subunits, does not occur. His-243 may therefore act as the basic residue that extracts the hydrogen of the alpha-carbon of Ser-68 in the ester in order to facilitate this reaction. The availability of the recombinant H243A S-adenosylmethionine decarboxylase proenzyme provides a useful model system to examine the processing reaction in vitro and test the design of specific inactivators aimed at blocking the production of the pyruvoyl prosthetic group.
Xiong
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Department of Cellular Physiology, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
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D012437:Adenosylmethionine Decarboxylase / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019811:Hydroxylamine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011773:Pyruvates / Q000737:chemistry; D013997:Time Factors
D004792:Enzyme Precursors; D004952:Esters; D011773:Pyruvates; D019811:Hydroxylamine; D012437:Adenosylmethionine Decarboxylase