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.
|Number of pages||6|
|Journal||Archives of Biochemistry and Biophysics|
|State||Published - Jul 1975|
Bibliographical noteFunding Information:
This work has supported by NIH Grant GM-17172.05 and by NSF Grant BMS75.08690. H. L.
ASJC Scopus subject areas
- Molecular Biology