A Proton Relaxation Rate Study of the Copper Analog of Escherichia coli Alkaline Phosphatase

Proton relaxation rates for the binary complex of the Cu2+ analog of Escherichia coli alkaline phosphatase and water have been measured. Titration of apoenzyme into copper salts shows approximately two tight copper-binding sites, Ki = 10-6 m, and 13 weak sites per enzyme dimer molecule. Enhancement of 1/T1p by a factor of 2 upon binding to enzyme is observed; this result for the first time suggests the participation of the metal in substrate binding. From the temperature dependence of T1p* and the fact that T1p g T2p it is concluded that T1p is not exchange-limited. From the frequency dependence of T1p and from the ratio of T1:T2 we estimate a correlation time, τc, of 4.6 x 10-9 s-1 which corresponds to the Cu2+-water proton distance r = 3.6 A when the number of ligand sites, q, = 1 or r = 3.2 when q = ½. The addition of phosphate has little effect on T1p for the copper enzyme. These results suggest that at least 1 water molecule or hydroxyl ion has access to the inner coordination sphere of the Cu2+ ion and that inorganic phosphate does not displace this ligand.