Kinetic Analysis of Arsenic−Metalation of Human Metallothionein: Significance of the Two-Domain Structure
journal contributionposted on 17.12.2008, 00:00 by Thanh T. Ngu, Alexandria Easton, Martin J. Stillman
Metallothionein (MT) is ubiquitous in Nature, underlying MT’s importance in the cellular chemistry of metals. Mammalian MT consists of two metal-binding domains while microorganisms like cyanobacteria consist of a single metal-binding domain MT. The evolution of a two-domain protein has been speculated on for some time; however, no conclusive evidence explaining the evolutionary necessity of the two-domain structure has been reported. The results presented in this report provide the complete kinetic analysis and subsequent mechanism of the As3+-metalation of the two-domain βαhMT and the isolated single domain fragments using time- and temperature-resolved electrospray ionization mass spectrometry. The mechanism for βαhMT binding As3+ is noncooperative and involves six sequential bimolecular reactions in which the α domain binds As3+ first followed by the β domain. At room temperature (295 K) and pH 3.5, the sequential individual rate constants, kn (n = 1−6) for the As3+-metalation of βαhMT starting at k1βα are 25, 24, 19, 14, 8.7, and 3.7 M−1s−1. The six rate constants follow an almost linear trend directly dependent on the number of unoccupied sites for the incoming metal. Analysis of the temperature-dependent kinetic electrospray ionization mass spectra data allowed determination of the activation energy for the formation of As1−H17-βαhMT (14 kJ mol−1) and As2−6-βαhMT (22 kJ mol−1). On the basis of the increased rate of metalation for the two-domain protein when compared with the isolated single-domain, we propose that there is an evolutionary advantage for the two-domain MT structures in higher organism, which allows MT to bind metals faster and, therefore, be a more efficient metal scavenger.