Publication
Biochemistry
Paper

Conformation States of Concanavalin A: Kinetics of Transitions Induced by Interaction with MN2+ and Ca2+

View publication

Abstract

Using measurements of the magnetic-field dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons over a wide range of field values (corresponding to proton Larmor frequencies from 0.01 to 50 MHz), we have investigated the interaction of Mn2+ and Ca2+ ions with concanavalin A (Con A) over the pH range 5.3 to 6.4, at 5 and 25 °C. Particular attention was given to time-dependent effects that occur upon addition or removal of metals. Limited amounts of Mn2+ added to solutions of apo-Con A bind at SI (the usual “transition-metal” site) to form a binary complex characterized by a large and pH-dependent dissociation constant, rapid exchange of Mn2+ ions with solvent, and a relatively large and pH-independent contribution to the proton relaxation rate. With S1 occupied, Ca2+ ions can bind at S2 (the usual “calcium-binding” site) to form a metastable ternary complex characterized by a relatively large and pH dependent dissociation constant for Ca2+ ions, rapid exchange of Ca2+ ions with solvent, and a relatively low and pH-independent contribution to the proton relaxation rate. We find that this metastable ternary complex undergoes a first-order transition to a stable ternary complex, with a pH-independent time constant of 17 ± 1 min at 5 °C and an activation energy of 22 kcal M-1. This stable ternary complex has the same relaxation contribution as the initial metastable complex, but differs in that the dissociation constant of Ca2+ is very low; the off-rate of both metals is of the order of days at 25 °C. Saccharide binding and agglutination studies are generally done with this form of Con A. We have also found that, in the absence of Ca2+, Mn2+ can bind at S2 as well as at S1 (S2 was previously thought to bind only Ca2+ and Cd2+) to form a metastable ternary complex which, like the metastable Mn2+-Ca2+-Con A complex, undergoes a transition to a stable state, but with a time constant that is much larger than for the Ca2+-containing ternary complex. In contrast to the stable Ca2+-Mn2+-Con A complex, the stable Mn2+-Con A ternary complex has a rather large dissociation constant, and the bound Mn2+ ions are in rapid equilibrium with solvent. The Mn2+ ions can be removed rapidly by the addition of ethylenediaminetetraacetic acid to produce apo-Con A in a metastable state that has different metal-binding properties than apo-Con A prepared by acid demetallization of the protein. Metals reintroduced to this metastable form of apo-Con A produce the stable ternary complexes with no observable time-dependent effects. This metastable form of apo-Con A reverts to its initial state after several days at 25 °C. We have fit the kinetic and thermodynamic data for the interaction of Mn2+ and Ca2+ ions with Con A with a model that postulates two conformation states for Con A that differ only slightly in their ground-state free energies, and are separated by an energy barrier of 22 kcal M-1. The conformation with the lower free energy is determined by the presence or absence of a metal ion at S2. The height of the energy barrier suggests that a cis-trans isomerization of a proline amide bond distinguishes the two conformations, implying that the difference between the conformations is in the secondary rather than the tertiary structure of the protein. © 1977, American Chemical Society. All rights reserved.

Date

01 May 2002

Publication

Biochemistry

Authors

Share