Reaction of Human Cd7metallothionein and N-Ethylmaleimide: Kinetic and Structural Insights from Electrospray Ionization Mass Spectrometry. Academic Article uri icon

abstract

  • The reaction of cadmium-binding human metallothionein-2A (CdMT) and N-ethylmaleimide (NEM) is investigated by electrospray ionization-ion mobility-mass spectrometry (ESI IM-MS). MS provides a direct measure of the distribution of the kinetic intermediates as the reaction proceeds and provides new insights into the relative kinetic stability of the individual metal-thiolate bonds in CdMT. The rate constants for the various metal-retaining intermediates (Cd(i), intermediate with i Cd ions attached) differ by >3 orders of magnitude: Cd< Cd< Cd< Cd Cd < Cd < Cd5. The reaction is viewed as a two-component cooperative process, rapid loss of three Cd ions followed by slow loss of the remaining four Cd ions, and CdNEMMT was observed as the least reactive intermediate during the entire displacement process. "MS-CID-IM-MS", a top-down approach that provides two-dimensional dispersion (size to charge by IM; mass to charge by MS) of the CID fragment ions, was used for direct analysis of the kinetic intermediate [CdNEMMT] ion. The results provide direct evidence that the four Cd ions located in the -domain are retained, indicative of the greater kinetic stability for the -domain. Further, the mapping of the alkylation sites in the [CdNEMMT] ion reveals that not only the nine cysteines in the -domain but Cys33 in the -domain is selectively labeled. The kinetic lability of the Cd-Cys33 bond is unexpected. The structural and functional implications of these findings are discussed.

published proceedings

  • Biochemistry

author list (cited authors)

  • Chen, S., & Russell, D. H.

citation count

  • 16

complete list of authors

  • Chen, Shu-Hua||Russell, David H

publication date

  • September 2015