Kinetic and Equilibrium Studies of Concanavalin A Activation by Calcium Ions

A. Dean Sherry, Margaret A. Lindorfer, Patrick Adams-Stemler, Oscar A. Milanes

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

The kinetics of conversion of concanavalin A (Con A) to the saccharide-binding conformation by Ca2+ alone were studied as a function of pH by monitoring the fluorescence quenching of 4-methylumbelliferyl -D-mannopyranoside. In the pH range 6.0-7.2, the protein conformational change associated with metal ion binding occurs upon binding of only one Ca2+ per P monomer. When sufficient Ca2+ is present to saturate the single Ca2+ site on P, the rate constant for the locking process, k2, is independent of pH between 6.4 and 7.2 and furthermore is identical within experimental error with k2 for Ca2+ in the presence of Mn2+. Equilibrium dialysis studies demonstrate that only one Ca2+ is bound in the final CPL conformation with a KD = 23 ± 5 at 25 °C. Ca2+ has been completely removed from CPL by gel filtration techniques, and the resulting metal-free PL structure has no measurable affinity for saccharides. The near-UV circular dichroism spectrum of CPL is identical with that of native Con A (CMPL) while the spectrum of PL is different from those of both P and CPL. The activation energy for the Ca2+-induced locking process is 14 kcal/mol at both pH 6.4 and pH 7.4 and is completely unaffected by the presence of Mn2+ at the higher pH. These data are consistent with a recent crystallographic report [Shoham, M., Yonath, A., Sussman, J. L, Moult, J., Traub, W., & Kalb, A. J. (1979) J. Mol. Biol. 131,137-155] of minor structural differences between native and demetalized Con A. We propose that Ca2+ binding at S2 produces a general ordering of the ligands at this site which in turn orders those side-chain residues involved in saccharide recognition.

Original languageEnglish (US)
Pages (from-to)3492-3499
Number of pages8
JournalBiochemistry
Volume20
Issue number12
DOIs
StatePublished - Jun 1981

ASJC Scopus subject areas

  • Biochemistry

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