Mechanism of substrate inactivation of Escherichia coli S-adenosylmethionine decarboxylase

Abstract

S-Adenosylmethionine decarboxylase, a pyruvoyl-containing decarboxylase, is inactivated in a time-dependent process under turnover conditions. The inactivation is dependent on the presence of both substrate and Mg2+, which is also required for enzyme activity. The rate of inactivation is dependent on the concentration of substrate and appears to be saturable. Inactivation by [methionyl-3,4-14C]-adenosylmethionine results in stoichiometric labeling of the protein. In contrast, when either S-[methyl-3H]adenosylmethionine or [8-14C]adenosylmethionine is used, there is virtually no incorporation of radioactivity. Automated Edman degradation of the alpha (pyruvoyl-containing) subunit reveals that substrate inactivation results in the conversion of the pyruvoyl group to an alanyl residue. These data suggest a mechanism of inactivation which involves the transamination of the nascent product to the pyruvoyl group, followed by the elimination of methylthioadenosine and the generation of a 2-propenal equivalent which could undergo a Michael addition to the enzyme. This is the first evidence for a transamination mechanism for substrate inactivation of a pyruvoyl enzyme.

In vivo mechanism-based inactivation of S -adenosylmethionine decarboxylases from Escherichia coli , Salmonella typhimurium , and Saccharomyces cerevisiae

“…As discussed further below, it seemed likely that the additional peaks that we found by MS of the isolated enzyme were due to in vivo modification similar to that reported by Anton and Kutny (16) and by Diaz and Anton (18) as occurring during the in vitro enzymatic decarboxylation of S-adenosylmethionine by the E. coli AdoMetDC. To determine whether the in vitro modification would result in the same additional peaks in the mass spectrometer, we incubated a sample of the enzyme that had little modification with AdoMet.…”

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.…”

“…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.…”

Catalytic Properties of the Archaeal S-Adenosylmethionine Decarboxylase from Methanococcus jannaschii

“…Schiff Base Trapping-Catalysis-dependent inactivation of AdoMet decarboxylase has been described both in vitro and in vivo and has been instructive in establishing a Schiff base mechanism (15, 17, 19, 21, 41 more complex mechanism involving modification of the catalytic cysteine after elimination of methylthioadenosine from the Schiff base (21,22,42). Mass spectrometry of proteins modified by the latter pathway revealed adducts with mass increases of ϩ57 and ϩ75 daltons resulting from the addition of CH 2 -CH 2 -CHO and the hydrate thereof (15).…”