ACTIVATION OF SACCHARIDE BINDING IN DE-METALIZED CONCANAVALIN-A BY TRANSITION-METAL IONS

Saccharide-binding activity is induced when apoconcanavalin A binds only one Mn2+ ion near pH 6.5 at 5 or 23 °C. The affinity of Mn2+-concanavalin A for 4-methylumbelliferyl α-d-mannopyranoside is about one-half that of Ca2+-concanavalin A under similar conditions. Equilibrium dialysis studies with 54Mn2+ show that a single Mn2+ binds per concanavalin A monomer in the presence and absence of saccharide. One Mn2+ is sufficient to activate sugar binding by each monomer. This result is similar to our earlier finding which showed that a single Ca2+ ion per protein monomer is most likely responsible for sugar-binding activation of Ca2+-concanavalin A near physiological pH [Christie, D. J., Alter, G. M., & Magnuson, J. A. (1978) Biochemistry 17, 4425]. Water proton relaxation rates for Mn2+-concanavalin A measured at 20.5 MHz, pH 6.4, and 23 αC decrease as the Mn2+ concentration is increased from 1 to 2 equiv of Mn2+ per monomer. This confirms similar observations made recently by others [Brown, R. D., III, Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883], No decrease in relaxation rates occurs when concanavalin A is incubated with Mn2+ at 5 αC. At least two different conformational states exist, one near 5 αC and one near 23 αC. The form at 5 αC converts, upon incubation at 23αC, to the higher temperature form. Both have full saccharide-binding activity, which only requires one bound Mn2+ per monomer. Mn2+ interaction with apoconcanavalin A is different than Ni2+, Co2+, and Zn2+ interaction. While binding with a stoichiometry of one metal ion per monomer, these other three transition metal ions do not induce saccharide-binding activity. Inactive Ni2+-concanavalin A, however, is activated in the presence of Mn2+, and no Ni2+ is displaced. This suggests that Mn2+ is not binding at exactly the same site as Ni2+, Co2+, and Zn2+, but may be at a hybrid site which overlaps in some way with both the specific SI transition metal ion site and the S2 Ca2+ ion site. © 1979, American Chemical Society. All rights reserved.