PROBING THE ROLE OF HISTIDINE-372 IN ZINC-BINDING AND THE CATALYTIC MECHANISM OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE BY SITE-SPECIFIC MUTAGENESIS

In the X-ray structure of Escherichia coli alkaline phosphatase at 2.0 -Angstrom resolution, His-372 was found only 3.8 Angstrom away from th e zinc and forms a hydrogen-bonding interaction with Asp-327, a bident ate ligand of the zinc at the M1 site. However, His-372 does not direc tly interact with the zinc atom at the M1 site. In order to investigat e the role of the side chain of His-372 in zinc binding and the cataly tic mechanism of Escherichia coil alkaline phosphatase, site-directed mutagenesis was used to convert His-372 to alanine. The fact that the His-372-->Ala enzyme has similar zinc binding affinity as the wild-typ e enzyme indicates that His-372 is not involved in zinc binding at the M1 site. However, the altered kinetic behavior of the mutant enzyme c ompared to the wild-type enzyme suggests that the imidazole ring of Hi s-372 plays an indirect role in the catalytic mechanism of the enzyme. The hydrolysis activity of the His-372-->Ala enzyme at pH 8.0 is 10-f old lower than that of the wild-type enzyme. In the presence of a phos phate acceptor at pH 8.0, the mutant enzyme is approximately 80% as ac tive as the wild-type enzyme. Therefore, the His-372-->Ala mutation se lectively enhances the transphosphorylation activity of the enzyme. Th e His-372-->Ala enzyme also exhibits 4- and 30-fold decreases in K-m a s compared to the wild-type enzyme in 0.1 M MOPS buffer and 1.0 M Tris , buffer at pH 8.0, respectively. A change in the rate-determining ste p at pH 8.0 is also observed for the His-372-->Ala enzyme compared wit h the wild-type enzyme. The presence of a transient burst in the pre-s teady-state kinetics of the His-372-->Ala enzyme indicates that breaki ng of the covalent phosphoserine bond is the rate-limiting step in the reaction. The lack of incorporation of [P-32]P-i into the His-372-->A la enzyme at pH 8.0 suggests the presence of a stable intermediate dur ing the transition from the enzyme-phosphate covalent complex to the e nzyme-phosphate noncovalent complex. Taken together, these kinetic res ults suggest that the hydroxyl group coordinated to the zinc atom at t he M1 site is a weaker nucleophile in the His-372-->Ala enzyme than in the wild-type enzyme. Therefore, the interaction between the side cha in of His-372 and Asp-327 may be important for stabilizing the zinc hy droxyl which is the nucleophilic group that is responsible for the bre akdown of the phosphoserine intermediate.