Interaction of calcium and manganese ions with apoconcanavalin A and sugar binding.
The interaction of apoconcanavalin A (apo-Con A) with Mn2+ and Ca2+ was studied at 25 degrees 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 x 103 M(-1) s(-1)); at higher pH the rate constant is greater than 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 x 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 greater than or equal to 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.
D001059:Apoproteins; D002118:Calcium; D003208:Concanavalin A; D005456:Fluorescent Dyes; D006027:Glycosides; D006923:Hymecromone / Q000031:analogs & derivatives; D007700:Kinetics; D008345:Manganese; D008362:Mannosides; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D001059:Apoproteins; D005456:Fluorescent Dyes; D006027:Glycosides; D008362:Mannosides; D003208:Concanavalin A; D006923:Hymecromone; D008345:Manganese; D002118:Calcium