Sugar binding properties of various metal ion induced conformations in concanavalin A

Abstract

Concanavalin A is known to undergo a first-order conformational transition when metals are added to the demetallized protein at pH 5.6 (Brown, R.D., III, et al. (1977) Biochemistry 16, 3883--3896). The rate constants for this process, which wer have measured using a polarographic technique, are identical when zinc, cobalt, or manganese occupies S1 and calcium occupies S2. The reducible sugar, p-nitrophenyl alpha-D-mannopyranoside, binds only to the locked conformational structure which is formed upon the addition of metals. The affinity of the protein for sugars is dependent upon occupancy of S1 and S2 and quite sensitive to the identity of the metal in S2. The metals may be removed from the locked protein structure and the protein temporarily retains its ability to bind with sugars but with a considerably lower affinity. The locked form of concanavalin A is unstable at a pH near 2 and unfolds to the unlocked structure with a half-life of 25 min resulting in simultaneous loss of metal and sugar binding.

Stoichiometry of magnesium and calcium ion binding to concanavalin A

“…A major distinction in the properties of the two conformations is that the locked form has a much greater affinity for metal ions and for saccharides. These conclusions are consistent with earlier stopped-flow NMR studies of Con A reported by Grimaldi & Sykes (1975) and have been confirmed by subsequent polarographic studies of the kinetics of binding of saccharide and a variety of metal ions to Con A by Sherry et al (1978) flow studies of metal ion and saccharide binding by Wilkins and co-workers (Harrington & Wilkins, 1978;Harrington et al, 1981), circular dichroism studies of metal ion substituted Con A (Cardin & Behnke, 1978), and U3Cd NMR studies of the binding of Cd2+ to Con A by Ellis and co-workers (Palmer et al, 1980). Koenig et al (1978) and Harrington & Wilkins (1978) showed that substantial saccharide binding activity of Con A was associated with the locked conformation even in the absence of bound metal ions.…”

“…In some cases (e.g., % and KCMP) values are known at several values of pH and temperature (Brown et al, 1977). Moreover, the rate constants that relate to some of the equilibrium constants (e.g., KLCMP and KLP) are also known (Brown et al, 1977;Harrington & Wilkins, 1978;Harrington et al, 1981;Sherry et al, 1978;Grimaldi & Sykes, 1975). Brown et al (1977) showed that Mn2+ alone could bind sequentially to SI and S2 to form a locked ternary complex.…”

Activation of saccharide binding in demetalized concanavalin A by transition metal ions

“…Under experimental conditions used in this present study, the other transition metal ions, Ni2+, Co2+, and Zn2+, cannot coordinate with the same ligands and induce activation. Other studies have recently appeared attempting to elucidate the role of divalent metal ions in the activation of saccharide binding with Con A (Koenig et al, 1978;Harrington & Wilkins, 1978; Sherry et al, 1978;Cardin & Behnke, 1978). Harrington & Wilkins (1978) conducted a kinetic study of Mn2+ and Ca2+ interaction with apo-Con A by following the fluorescence quenching of MUM.…”

Interaction of calcium and manganese ions with apoconcanavalin A and sugar binding

“…After this manuscript was submitted, Sherry et al (1978) reported first-order conformational rate constants (corresponding to k) using PNM and polarography. Binding ability to PNM of the apoprotein formed by adding EDTA to metal forms of Con A was also demonstrated.…”