Kinetics of binding of Co²⁺ to apoalkaline phosphatase from Escherichia coli

The kinetics of the binding of Co²⁺ to apoalkaline phosphatase from Escherichia coli have been followed by stopped flow spectroscopy of the tyrosines and bound Co²⁺, revealing three well-separated kinetic phases with time constants in the milliseconds, seconds, and minutes time ranges. Four Co²⁺ are bound per dimer judging from the saturation behavior of an isomerization reaction linked to a rapid metal binding step. These Co²⁺ are distributed in pairs to each of two different types of binding sites distinguishable by their participation in two different isomerization-linked transients in the seconds and minutes time ranges, respectively. Two of the observed isomerizations are tentatively assigned to subtle protein conformational changes since they are reflected in both the Co²⁺ and aromatic amino acid chromophores but not by changes in ORD, CD, viscosity, or ultracentrifugation properties. They may therefore represent local changes in regions of the protein near the metal sites.