title: The zinc balance: competitive zinc metalation of carbonic anhydrase and metallothionein 1A.
abstract: The small, cysteine-rich metallothionein family of proteins is currently considered to play a critical role in the provision of metals to metalloenzymes. However, there is limited information available on the mechanisms of these fundamentally important interactions. We report on the competitive zinc metalation of apocarbonic anhydrase in the presence of apometallothionein 1A using electrospray-ionization mass spectrometry. These experiments revealed the relative affinities of zinc to all species in solution. The carbonic anhydrase is shown to compete efficiently only against Zn5-7MT. The calculated equilibrium zinc binding constants of each of the 7 zinc metallothionein 1A species ranged from a high of (log(KF)) 12.5 to a low of 11.8. The 8 equilibrium constants connecting the 10 active species in competition for the zinc were modeled by fitting the KF values of the 8 competitive bimolecular reactions to the ESI-mass spectral data. These modeled K values are shown to be experimentally connected to the metalation efficiency of the carbonic anhydrase. The series of 7 metallothionein binding affinities for zinc highlight the buffering role of zinc metallothioneins that permit simultaneously zinc storage and zinc sensing. Finally, the significance of the multiple zinc binding affinities of zinc metallothionein is discussed in relation to zinc homeostasis.
authors.affiliation: Department of Chemistry and ‡Department of Biology, The University of Western Ontario , London, Ontario, Canada N6A 5B7.
authors.forename: Tyler B J
authors.initials: TB
authors.lastname: Pinter
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stillman
mesh_terms: D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001051:Apoenzymes; D001059:Apoproteins; C530099:MT1A protein, human; D011994:Recombinant Proteins; D008668:Metallothionein; D002256:Carbonic Anhydrases; D015032:Zinc