3-D STRUCTURE OF A MUTANT (ASP101-]SER) OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE WITH HIGHER CATALYTIC ACTIVITY

Mutagenesis of the absolutely conserved residue Asp101 of the non-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1) from E. coli has produced an enzyme with increased k(cat). The carboxyl group of the Asp101 residue has been proposed to be involved in the positioning of Arg166 and the formation of the helix that contains the active site Ser102. The crystal structure of the Asp101 --> Ser mutant has been refined at 2.5 angstrom to a final crystallographic R-factor of 0.173. The altered active site structure of the mutant is compared with that of the wild-type as well as with the structures of the mutant enzyme soaked in two known alkaline phosphatase inhibitors (inorganic phosphate and arsenate). The changes affect primarily the side chain of Arg166 which, by losing the hydrogen bond interaction with the carboxyl side chain of Asp101, becomes more flexible. This analysis, in conjunction with product inhibition studies of the mutant enzyme, suggests that at high pH (> 7) the enzyme achieves a quicker catalytic turnover by allowing a faster release of the product.