Kinetics of calcium binding to calbindin mutants

Abstract

The kinetics of calcium dissociation from wild‐type bovine calbindin D9k (the smallest protein known with a pair of EF‐hand calcium‐binding sites) and five mutants with single amino‐acid substitutions and/or deletions has been studied by stopped‐flow fluorescence methods, using the calcium chelator Quin 2. The modifications are confined to the N‐terminal half of the molecule, at or near the first calcium‐binding site (I). Substitutions and deletions of amino acids in the calcium‐binding loop of site I primarily affect the rate of Ca2+ dissociation from this site with only minor effects on the dynamic properties of the C‐terminal calcium‐binding site (II). This finding corroborates and extends previous kinetic results obtained from 43Ca‐NMR studies on the same set of mutants. By contrast, removal of the hydrogen bond between Tyr‐13 and Glu‐35, an interaction linking the two α‐helices flanking site I, through replacement of Tyr‐13 with Phe, has no observable effect on the rate of Ca2+ dissociation from the protein. Comparison of this kinetic data with binding‐constant data, previously obtained in our laboratories, shows that the decrease in Ca2+‐affinity of site I, observed in most mutants, is predominantly due to an increased off‐rate from this site. At low ionic strengths the second‐order rate constants for Ca2+‐binding to both Ca2+ sites of calbindin D9k are calculated to be of the order of 109 M−1 s−1 for all proteins studied. At higher ionic strengths (0.1 M KCl) the rates of Ca2+ dissociation from both sites are increased by a factor of three or more, suggesting a transition state which is ionic in nature. Copyright © 1988, Wiley Blackwell. All rights reserved