Reversible alkylation of an active site methionine residue in dehydroquinase.

Iodoacetic acid inactivates dehydroquinase by simultaneously alkylating 2 methionine residues (Met-23 and Met-205), presumed to be active site residues (described in Kleanthous, C., Campbell, D. G., and Coggins, J. R. (1990) J. Biol. Chem. 265, 10929-10934). Although both sites are carboxymethylated to the same degree in the inactivated enzyme, the modification of Met-205 may be reversed by treatment with mercaptoethanol at alkaline pH, as shown by the stoichiometric loss of label from this site. This, in turn, leads to partial reactivation of the inactive enzyme. Alkylation of Met-23 is not reversible under these conditions. The chemistry of the cleavage reaction at Met-205 was investigated by isolating the cleavage product which was identified by mass spectrometry as the ammonium salt of 2-hydroxyethyl thioacetate. This result is consistent with nucleophilic attack by the thiolate anion of mercaptoethanol on the alpha-carbon of the carboxymethyl moiety, which restores the side chain of the methionine residue (Met-205) and liberates 2-hydroxyethyl thioacetate. The differential reactivity of the 2 carboxymethylated methionine residues toward mercaptoethanol is likely to be a reflection of their different microenvironments in the folded protein. This assertion is borne out by unfolding experiments which indicate that neither of the carboxymethylated methionine residues in dicarboxymethylated dehydroquinase is susceptible to mercaptoethanol cleavage if the protein is first denatured by either guanidine hydrochloride or urea. Furthermore, this denatured material refolds after removal of denaturant to yield protein with reactivation properties similar to untreated, dicarboxymethylated enzyme.