Zn2+ and Co2+-alkaline phosphatases of E. coli. A comparative kinetic study.

1 For the Zn2+-phosphatase, the ratio k3/k2 (pH 8.4) of the dephosphorylation (k3) and phosphorylation (k2) rates is very similar, about 1.4–1.7, for substrates which are very different chemically such as p-nitrophenyl phosphate, β-glycerophosphate, 5′-AMP and ATP. This means that k2 is nearly the same for all these substrates. On the other hand with o-carboxyphenyl phosphate, phosphorylation is the determining step. 2 The Co2+-enzyme has a much lower specific activity than the Zn2+-phosphatase. The substitution of Zn2+ by Co2+ decreased the rate of phosphorylation by a factor of 6.4 for o-carboxyphenyl phosphate and 16 for the above mentioned substrates; no such decrease is observed on k3. The phosphorylation is inhibited by nucleophilic agents which otherwise activate the Zn2+-enzyme in the same conditions. The Co2+-phosphatase offers a unique opportunity to compare the free energy of binding of the different substrates to form the Michaelis complex; those free energies are strikingly similar, they vary between – 6.1 and – 7.0 kcal. This is further evidence that specificity of the enzyme is not directed against the R part of RO PO3 even in such a peculiar case as ATP. Neither the Zn2+ nor the Co2+-enzyme are able to catalyse the hydrolysis of a P-N bond. Organic phosphates (P-O bond) bind better to the phosphatase than p-chloro-anilido-phosphonic acid (P-N bond). The exchange of Zn2+ by Co2+ decrease only slightly the affinity for the active center of organic substrates or inhibitors; a factor of 1.78 for o-carboxyphenyl phosphate and 2.65 for p-chloroanilido-phosphonic acid. The difference is more pronounced for inorganic phosphate; a factor of 23 (pH 9.16) and 27 (pH 8.5). 3 Two ionisable groups are important for the binding of the substrate to the active center. One must be in the basic form, it has a pK of 7.1 and 7.4 in the Zn2+ and Co2+-phosphatase, respectively. The other must be acidic, the corresponding pK values are 8.6 and 8.9, respectively. With both metallo-enzymes the phosphorylation step is pH-independent between pH 6 and 9. The dephosphorylation step is dependent upon the ionization of a group which must be in the basic form and has a pK of 7.5 for the Zn2+-phosphatase. 4 The enzyme remains dimeric in the course of the catalysis and it seems that there is only one active site phosphorylated per dimer. k2 and k3 are decreased in 2H2O indicating possibly rate-determining proton transfers. The enthalpy and entropy of activation corresponding to both steps have been calculated.