Role of second metal ion in establishing active conformations of concanavalin A

Abstract

The stoichiometry of Mn2+ binding to concanavalin A was found to be influenced by temperature, pH, and the presence or absence of saccharide. Demetalized concanavalin A binds one Mn2+ (S1 site) at 5.degree. C, pH 6.5, and two Mn2+ at 25.degree. C (S1 and S2 sites). The association constants for Mn2+ are 6.2 .times. 105 and 3.7 .times. 104 M-1 for the S1 and S2 sites, respectively, at 25.degree. C. Concanavalin A with one Mn2+ bound per monomer remains in an open conformation and exhibits a relatively high water protein relaxation rate. Concanavalin A with two Mn2+ ions remains in a closed conformation characterized by a lower relaxation rate. The rate of binding of the second Mn2+ to concanavalin A was determined by ESR and the rate of conversion of open form to closed form (folding over) as determined by proton relaxation rate measurements gave an identical rate constant of 80.0 .+-. 5.8 M-1 h-1 at 17.degree. C. Ca2+, Sr2+, and high levels of methyl .alpha.-D-mannopyranoside also induce folding of concanavalin A. Ca2+ is not catalytic but stoichiometric in causing the folding. Mn2+ in the S1 site can be displaced by Ni2+, Co2+, and Zn2+, and Mn2+ in the S2 site can be displaced by Ca2+ and Sr2+. Concanavalin A with Ni2+, Co2+, Zn2+, or Mn2+ in the S1 site and Ca2+ or Sr2+ in the S2 site has a higher affinity for methylumbelliferyl .alpha.-D-mannopyranoside than Ni-Mn-, Co-Mn-, Zn-Mn-, and Cd-Cd-concanavalin A.