title: Reaction of Human Cd7metallothionein and N-Ethylmaleimide: Kinetic and Structural Insights from Electrospray Ionization Mass Spectrometry.
abstract: The reaction of cadmium-binding human metallothionein-2A (Cd₇MT) 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 Cd₇MT. The rate constants for the various metal-retaining intermediates (Cd(i), intermediate with i Cd²⁺ ions attached) differ by >3 orders of magnitude: Cd₄< Cd₃< Cd₂< Cd₁∼ Cd₆ < Cd₇ < Cd5. The reaction is viewed as a two-component cooperative process, rapid loss of three Cd²⁺ ions followed by slow loss of the remaining four Cd²⁺ ions, and Cd₄NEM₁₀MT 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 [Cd₄NEM₁₀MT]⁵⁺ ion. The results provide direct evidence that the four Cd²⁺ ions located in the α-domain are retained, indicative of the greater kinetic stability for the α-domain. Further, the mapping of the alkylation sites in the [Cd₄NEM₁₀MT]⁵⁺ ion reveals that not only the nine cysteines in the β-domain but Cys33 in the α-domain is selectively labeled. The kinetic lability of the Cd-Cys33 bond is unexpected. The structural and functional implications of these findings are discussed.
authors.affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Shu-Hua
authors.initials: SH
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: David H
authors.initials: DH
authors.lastname: Russell
mesh_terms: D002104:Cadmium / Q000737:chemistry*; D005033:Ethylmaleimide / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: C491053:MT2A protein, human; D002104:Cadmium; D008668:Metallothionein; D005033:Ethylmaleimide