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.