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

Abstract

The interaction of [jack bean] apoconcanavalin A (apo-Con A) with Mn2+ and Ca2+ was studied at 25.degree. C using fluorescence stopped flow. The reaction was monitored using 4-methylumbelliferyl .alpha.-D-mannopyranoside whose fluorescence is quenched on binding to the metalloproteins. At pH 5.0, entry of Mn2+ into apo is second-order (rate constant = 1.2 .times. 103 M-1 s-1); at higher pH the rate constant is > 104 M-1 s-1. Reaction of excess Ca2+ with Mn(Con A) is pseudo-first-order with kobsd = Kk [Ca2+](1 + K [Ca2+)-1. This is interpreted as rapid formation of unlocked MnCa(Con A), with a formation constant K = 3.5 .times. 102 M-1, which transforms (k = 0.026 s-1) to a locked form, indistinguishable from native. At pH 6.4 and 7.2, K .gtoreq. 103 M-1 and k = 0.043 and 0.050 s-1, respectively. Ca(Con A) and Mn(Con A) precipitate glycogen and bind to 4-methylumbelliferyl .alpha.-D-mannopyranoside as effectively as native protein at pH 7.2. Treatment of the Ca or Mn forms with EDTA produces an apo form with a small binding capacity, which it loses slowly.