Why Zinc II and Serine in Alkaline Phosphatase

Alkaline phosphatase (AP) is a (Znn)2-containing phosphomonoester-ase that hydrolyzes phosphomonoesters (RO—POf-) at alkaline pH (7). Ser102 under the influence of one of the zinc(II) ions at the active center 1 (Fig. 2) is directly involved in phosphate hydrolysis (8). On the basis of X-ray structure and NMR studies (9), the mechanism now accepted is that the phosphate substrate, initially recognized by cooperative 

A comparison of NA hydrolysis by 8 and 9 shows for the first time that Znu-bound alkoxides may lead to better nucleophiles than Zn11-bound hydroxides in an aqueous environment. Unless the alkoxide anion is bound to zinc(II) ion, it may rather work as a general base to yield hydroxide, which then yields the active nucleophile. Hence, zinc(II) ion may be viewed as an alkoxide-protecting agent. It is of 

The labile character of the Zn11— OR species 8b (although isolable as crystals, 8b is stable only at a limited alkaline pH region in aqueous solution) and the vulnerability of the acyl intermediate 10 in aqueous solution prevented more extensive, quantitative studies about the nature of Znn-bound alkoxide. 

The potentiometric pH titration disclosed monodeprotonation with a pK value of 7.6 at 25°C. On the basis of NMR spectroscopic and anionbinding studies of 13, the monodeprotonated species was assigned to the OH -bound ZnL complex 14, rather than the pendent alkoxide complex as seen with [12]aneN3 8b. Nevertheless, among all past zinc(II) complexes, 14 seems to be the most active catalyst for NA hydrolysis. From kinetic studies in 10% (v/v) CH3CN at 25°C and pH 

5 with I = 0.1, where the zinc(II) complex remains stable either as 13 or 14, the rate-pH profile gave a sigmoidal relationship with an inflection point (i.e., kinetic pKa) at pH 7.7, which almost corresponds to the pKa value for the 13 14 equilibrium (see Fig. 3). The second-order (first-order each in [14] and [NA]) rate constant of 0.46 M 1sec 1 is ca. 10 times greater than the corresponding value of 4.7 x 2 M 1 sec-1 for a reference iV-methylcyclen-Zn —OH 15b (pAa for 15a 15b is 7.6 at 25°C with 7 = 0.1) (19). Furthermore, NA is hydrolyzed 

The reaction of 14 may remind one of the well-established reaction mechanism for chymotrypsin (Fig. 5) (20). By comparing the acyl-trans-fer reaction of complex 14 with that of chymotrypsin 17, we find that the alcoholic nucleophiles in 14 and 17 are activated by Zn —OH and imidazole (in a triad), respectively. Several common features should be pointed out (i) Both reactions proceed via two-step reaction (i.e., double displacement), (ii) The basicity of Zn —OH (pK = 7.7) is somewhat similar to that of imidazole (pK = ca. 7). (iii) The initial acyl-transfer reactions to alcoholic OH groups are rate determining, (iv) In NA hydrolysis with chymotrypsin, the pH dependence of both the acylation (17 18) and the deacylation (19 — 17) steps point to 

Comparison of Second-Order Rate Constants (M sec ) in Hydrolysis (or Acetyl and Phosphoryl Transfer) of 4-Nitrophenyl Acetate, Methyl Acetate and Bis(4-nitrophenyl) Phosphate 

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