Kinetics of calcium binding to calbindin mutants.
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 alpha-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 10(9) 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.
Forsén
S
S
Chemical Centre, Lund University, Sweden.
Linse
S
S
Thulin
E
E
Lindegård
B
B
Martin
S R
SR
Bayley
P M
PM
Brodin
P
P
Grundström
T
T
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D009154:Mutation; D064030:S100 Calcium Binding Protein G / Q000235:genetics* / Q000378:metabolism
D000596:Amino Acids; D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D002118:Calcium