Kinetics of metal binding by a zinc finger peptide

The kinetics of cobalt(II) and zinc(II) binding by a prototypical zinc finger peptide has been examined by stopped-flow spectrometry. Cobalt(II) binding was demonstrated to be second order, first order in each peptide and cobalt(II) concentration. At pH 6.55 in 100 mM HEPES, 100 mM NaCl buffer the second order rate constant was 7.5(+/- 2.1) x 10(4) M-1 s(-1). The displacement of cobalt(II) by zinc(II) was examined at pH 7.0 as a function of both cobalt(II) and zinc(II) concentration. The data are consistent with a dissociative mechanism with the rate of zinc(II) binding being substantially faster than cobalt(II) binding. The rate constant for cobalt(II) dissociation was 5.3(+/- 0.5) x 10(-2) s(-1). The ratio of the rate constants for zinc(II) and cobalt(II) binding was found to be 66 +/- 3, indicating that the rate constant for zinc(II) is 2.8(+/- 0.4) x 10(7) M-1 s(-1) at pH 7.0. This indicates that the rate of zinc(II) dissociation is of 1.6(+/- 0.6) x 10(-4) s(-1). The magnitude of the kinetic preference for zinc(TI) over cobalt(II) revealed in metal binding rate constants is observed for many other zinc metalloproteins despite the fact that these rate constants vary over eight orders of magnitude. (C) 2000 Elsevier Science S.A. All rights reserved.