Role of Cysteine-82 in the Catalytic Mechanism of Human S-Adenosylmethionine Decarboxylase

Abstract

S-Adenosylmethionine decarboxylase is a pyruvate-dependent enzyme. The enzyme forms a Schiff base with substrate, S-adenosylmethionine, through the pyruvoyl moiety. This facilitates the release of CO2 from the substrate, which must then be protonated on the alpha carbon in order to permit hydrolysis of the Schiff base to release the product. The catalytic mechanism of human S-adenosylmethionine decarboxylase was investigated via mutagenic and kinetic approaches. The results of enzyme kinetic studies indicated that Cys-82 is a crucial residue for activity and this residue has a basic pKa. Iodoacetic acid inhibited wild-type enzyme activity in a time- and pH-dependent manner but did not affect the already reduced activity of mutant C82A. Reaction of this mutant with iodoacetic acid led to approximately one less mole of reagent being incorporated per mole of enzyme alphabeta dimer than with wild-type S-adenosylmethionine decarboxylase. Both wild-type and C82A mutant S-adenosylmethionine decarboxylases were inactivated by substrate-mediated transamination, but this reaction occurred much more frequently with C82A than with wild-type enzyme. A major proportion of the recombinant C82A mutant protein was in the transaminated form in which the pyruvoyl cofactor is converted into alanine. This suggests that incorrect protonation of the pyruvate, rather than the substrate, occurs much more readily when Cys-82 is altered. On the basis of these results, it was postulated that residue Cys-82 may be the proton donor of the decarboxylation reaction catalyzed by S-adenosylmethionine decarboxylase.

S-Adenosylmethionine Decarboxylase Degradation by the 26 S Proteasome Is Accelerated by Substrate-mediated Transamination

“…Although inactivation because of an initial small (non-26 S proteasome) proteolytic clip cannot be completely ruled out by the current results, the accumulated evidence from our work and that from other laboratories (22,23,36) points to a substrate-mediated transamination of the covalently linked pyruvoyl cofactor (and possibly additional accompanying alkylation events) as the cause of the observed inactivation. Taken together, these results strongly suggest that the enzyme may be transaminated before complete degradation by the 26 S proteasome and that such transamination may serve as a trigger for accelerated degradation.…”

“…Plasmids Used-The His-tagged human AdoMetDC cDNA portion of the previously produced bacterial expression construct pQE-HISSAMI (22) was subcloned into the mammalian expression vector pcDNA3.1/Zeo(-) (Invitrogen) using PstI and XhoI restriction enzyme cuts on both the vector and original construct. Equal amounts of the empty pcDNA3.1/Zeo(-) vector were used as a transfection control for the His-tagged AdoMetDC construct.…”

“…The increased antigen amounts ob- With the exception of substrate AdoMet-mediated transamination and related alkylations (22, 23, 36) and a suggestion of a modified residue in bovine AdoMetDC (37), no other posttranslational modifications of mature (cleaved) AdoMetDC have been described. Transamination by either the substrate AdoMet or the catalytic product dcAdoMet converts the covalently linked pyruvoyl cofactor to alanine and permanently inactivates AdoMetDC (22,23), which has led to the suggestion that the rapid turnover of AdoMetDC may be teleologically explained by the need to remove this constantly forming pool of inactive enzyme (5). The intracellular concentration of AdoMet is below the K m for the AdoMetDC enzyme, so an increase in the substrate concentration would increase the number of catalytic turnovers proportionally.…”

“…In addition to loss of AdoMetDC activity through degradation of the enzyme (loss of antigenic AdoMetDC), it is also known that AdoMetDC activity can be lost by permanent covalent inactivation of the enzyme in a process known as substrate-mediated transamination. Transamination transfers the amine group from the substrate AdoMet, converting the covalently linked pyruvoyl cofactor to an alanine (22,23). The depletion of intracellular AdoMet by the presence of the AdoMet synthase inhibitor L-2-amino-4-methoxy-cis-but-3-enoic acid has been reported to lengthen the half-life of AdoMetDC activity loss 3-fold (15), suggesting that the rate of this inactivation might be related to the intracellular concentrations of the substrate AdoMet, al-though direct stabilization effects of the L-cisAMB could not be completely ruled out in those experiments.…”

Structural Roles of Cysteine 50 and Cysteine 230 Residues in Arabidopsis thaliana S-Adenosylmethionine Decarboxylase

“…This includes the sequences (KTCGTT) that contain an essential active site lysine and cysteine residues (Stanley and Pegg, 1991;Stanley et al, 1994;Xiong et al, 1997). In spite of rather extensive studies on the functionally important residues of AdoMetDC, almost all of the studies were confined to the enzyme active site (Stanley and Pegg, 1991;Stanley et al, 1994;Xiong et al, 1999), except the essential Cys 83 residue in the active site. There are presently no studies on the other conserved cysteine residues of plant AdoMetDC.…”