ROLES OF ZINC(II) ION IN PHOSPHATASES - A MODEL STUDY WITH ZINC(II) MACROCYCLIC POLYAMINE COMPLEXES

The zinc(II) complexes of 1,5,9-triazacyclododecane (L4, [12]aneN3) and 1,4,7,10-tetraazacyclododecane (L2, [12]aneN4) promote the hydrolysis of tris(4-nitrophenyl) phosphate, TNP0 (a neutral phosphotriester), and bis(4-nitrophenyl) phosphate, BNP- (a monoanionic phosphodiester). Kinetic studies show that the reactive species are commonly L-Zn(II)-OH- and the kinetically obtained pK(a) values are almost identical to those obtained thermodynamically (pK(a)(H20)). The comparative reactivities of OH-, L4-Zn(II)-OH-, and L2-Zn(II)-OH- with different pK(a)(H2O) values of 15.7, 7.3, and 8.0 have been studied to understand the role of Zn(II) in alkaline phosphatases. For the neutral phosphotriester TNP0, the second-order rate constants (M-1 s-1) at 25-degrees-C and I = 0.20 are 10.7, 7.0, and 3.7, respectively, indicating free OH- ion (the most basic OH- species) is the best promoter. On the other hand, toward the anionic phosphodiester BNP-, the L4-Zn(II)-OH- becomes more efficient than free OH- ion: The second-order rate constants (X 10(5) M-1 s-1) are 2.4, 8.5, and 2.1, respectively, at 35-degrees-C and I = 0.20. A bifunctional ("hybrid") mechanism of Zn(II) (especially the Zn(II)-L4 system) toward phosphates (especially the anionic phosphate), in which the Zn(II)-bound OH- acts as a nucleophile, while the vacant Zn(II) coordination site anchors the substrate P = O or P - O- group, is well disclosed by comparing these kinetic results with the second-order rate constants (at 25-degrees-C and I = 0.10) of 4-nitrophenyl acetate hydrolysis, 9.5, 4.1 X 10(-2), and 1.1 X 10(-1) M-1 s-1, respectively, where all of the OH- species act merely as nucleophiles to the carbonyl group, as indicated by a linear relationship between the pK(a)(H2O) values and the rate constants. Phosphate anion affinity constants K(A-) (M-1) with the Zn(II)-L4 complex determined by pH titration are 10(3.5) for phenyl phosphate, PP2-, and 10(3.1) for 4-nitrophenyl phosphate, NP2-, which are ca. 25 times larger than those with the less acidic Zn(II)aq ion.