KINETIC STUDY OF MANGANESE ASSOCIATION TO DE-METALLIZED CONCANAVALIN-A

The saccharide binding properties of the mitogenic lectin concanavalin A are linked to the association of two metals, Mn2+ and Ca2+. We have examined the binding properties of these metals with the specific purpose of looking for interactions between them. In a previous publication [Alter et al. (1977) Biochemistry 16, 4034] we reported that preincubation with Ca2+ of demetallized concanavalin A changes Mn2+ binding from a noncooperative to a cooperative process. In an extention of these studies, we now report that preincubation with Ca2+ of demetallized concanavalin A has a profound effect on the kinetics of Mn2+ binding. Using electron spin resonance and proton relaxation enhancement methods to examine both the time course of Mn2+ binding and changes in the environment of bound Mn, we have found that Ca2+ slows Mn2+ association relative to the rate in the absence of Ca2+. Time courses obtained upon addition of Mn2+ to Ca2+-preincubated, demetallized concanavalin A are consistent with a rate-determining step involving binding of Mn2+ to the second manganese site of a concanavalin A dimer. Further, the pH dependence of the association rate indicates that two groups with pAT values of approximately 5.3 and 6.0 influence the rate of this metal's association. Activation parameters for the process at pH 5.1 and 6.45 are also reported. Results of this kinetic study are combined with the previously reported equilibrium studies to propose a scheme for Mn2+ binding to Ca2+-preincubated concanavalin A in which the rate-limiting step for the association process is the binding of a second Mn2+ to concanavalin A dimers. © 1979, American Chemical Society. All rights reserved.