Kinetics of reversible N-ethylmaleimide alkylation of metallothionein and the subsequent metal release

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• 作者：C. F. Shaw III. ; Libin He ; Amalia Mu?oz ; M. Meral Savas ; Susan Chi ; Cynthia L. Fink ; Tong Gan and David H. Petering
• 关键词：Key words  ; Metallothionein ; N ; Ethylmaleimide ; Alkylation ; Kinetics and mechanism ; 111Cd ; NMR
• 刊名：Journal of Biological Inorganic Chemistry
• 出版年：1997
• 出版时间：February 1997
• 年：1997
• 卷：2
• 期：1
• 页码：65-73
• 全文大小：242 KB

文摘

 The model alkylating agent N-ethylmaleimide (NEM) reacts reversibly at the metal-bound thiolates of Zn₇MT and Cd₇MT. An unprecedented feature of this reaction is that it approaches equilibrium and requires a large excess of NEM (>1 mM for 3 μM protein) to drive it to completion. The complex kinetics of the reaction can be followed by monitoring the release of bound metal ions using the metallochromic dyes Zincon (ZI) for Zn₇MT and pyridylazoresorcinol for Cd₇MT. An initial lag phase is followed by more rapid release of zinc ions. The observed pseudo-first-order rate constants for the two phases are independent of the ZI and Zn₇MT concentrations. The complex NEM concentration dependence of each phase, k _f, obs=k f ₁+k f ₂ [NEM] and k _s, obs=k s ₁+k s ₂ [NEM],