KINETICS OF REVERSIBLE N-ETHYLMALEIMIDE ALKYLATION OF METALLOTHIONEINAND THE SUBSEQUENT METAL RELEASE

The model alkylating agent N-ethylmaleimide (NEM) reacts reversibly at the metal-bound thiolates of Zn(7)MT and Cd(7)MT. An unprecedented fe ature of this reaction is that it approaches equilibrium and requires a large excess of NEM (>1 mM for 3 mu M protein) to drive it to comple tion. The complex kinetics of the reaction can be followed by monitori ng the release of bound metal ions using the metallochromic dyes Zinco n (ZI) for Zn(7)MT and pyridylazoresorcinol for Cd(7)MT. An initial la g phase is followed by more rapid release of zinc ions. The observed p seudo-first-order rate constants for the two phases are independent of the ZI and Zn(7)MT concentrations. The complex NEM concentration depe ndence of each phase, k(f,obs) = k(1)(f) + k(2)(f) [NEM] and k(s,obs) = k(1)(s) + k(2)(s) [NEM], demonstrates that the forward reactions are second order and the reverse reactions are first order. The alkylatio n can be reversed using 2-mercaptoethanol to compete for the protein-b ound NEM and regenerate the Zn-binding capability of alkylated MT. An explanation of these observations, based on the reversibility of cyste ine alkylation by NEM, was developed and tested. The reactions of Cd(7 )MT are less complete than those of Zn(7)MT and occur more slowly. Cd- 111-NMR studies of the partially alkylated (111)Cd(7)MT reveal that re action with only four equivalents of NEM completely alters the cluster structure and eliminates the spectral signatures of the alpha and bet a clusters, although very little cadmium has been removed from the pro tein. This finding substantiates the proposed kinetic intermediate, a partially alkylated MT with complete or nearly complete retention of t he metal ions, and rules out the possibility of cooperative reactions at either cluster.