Diamagnetic bivalent metal ion NMR studies of metalloproteins;Zn 2+Insulin and Zn 2+-concanavalin complexes

Abstract

Diamagnetic bivalent metal ions such as Mg 2+, Ca 2a, and Zn 2+ are indispensable for the full activity of metalloenzymes, but are non-chromophoric. Thus, paramagnetic and chromophoric metal ions such as Co 2+, Mn 2+, and Tb 3+ were substituted for Mg 2+, Ca 2+, or Zn 2+ in the metalloenzymes and have been thought to be useful as a mimetic probe to the diamagnetic bivalent metal ions. However, the enzymatic activities and structures of the metal-binding sites in the metal substituted enzymes might be different from the native enzymes, and then it is necesary to analyze directly the structural role of the divalent ions such as Mg 2+, Ca 2+, and Zn 2+ in the metalloenzymes. Thus, metal ion NMR has shown in studying the structure of the metalloenzymes, especially the metal-binding site, the ligating candidates, or the motional behavior of the bound metal ions. In succession to our bivalent diamagnetic metal ion NMR studies [1–5], we would like to present 67Zn NMR studies on Zn 2+-insulin and Zn 2+-concanavalin A complexes in this paper. NMR characteristics of 67Zn (I =5/2) are similar to those of 25Mg (I = 5/2) and 43Ca(I = 7/2), since all the nuclei a guadrupole moment. However, 67Zn NMR spectra of aqueous Zn 2+ are different from 25Mg and 43Ca NMR spectra of aqueous Mg 2+ and Ca 2+ - 67Zn NMR spectra of aqueous Zn 2+ have a marked concentration dependence in terms of the half-band widths compared with those of 25Mg and 43Ca NMR of aqueous Mg 2+ and Ca 2+ [1–3]. For example, ZnCl 2 (2 M), pH 4.0, exhibited a very broad 67Zn NMR with a half-band width of 170 Hz, and dilution of ZnCl 2 solution to 50 m M ▪ led to a narrower 67Zn NMR with a half-band width of 12 Hz. By adding 1m M bovine insulin to the 50 m M ZnCl 2 solution, at pH 2.95, the half-band width of 67Zn NMR increased by three times. Addition of insulin more than 1 m M led to a broader 67Zn NMR. The determined T 1/T 2 values of the 2 M and 50 m M ZnCl 2 solutions are almost unity. But, the T 1/T 2 ratio of 67Zn NMR of the Zn 2+-insulin complex is not unity (1.36). From these findings, the correlation time, τ c and the quadrupole coupling constant are evaluated. The halfband widths of 67Zn NMR are temperature dependent, when Zn 2+ is bound to polymeric ligands. It is suggested from the temperature dependence that molecular weights of the ligands are closely correlated with the mechanism of the 67Zn NMR of the Zn 2+ complexes. The relaxation mechanism on the 67Zn NMR will be discussed. Scheme 1. Proposed kinetic mechanism of carbonic anhydrase. H to the right of E represents protonated catalytic group (zinc-bound H 2O). H to the left of E represents protonated His-64.