Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase

ABSTRACT: The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the k,, for the mutant enzymes decreases by approximately 30-fold while the K, increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the k,,, for the mutant enzymes is reduced by less than 3-fold, while the K, increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the k, , /K, ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166- Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F. J. M., & Riordan, J. F. (1974) Biochemistry 13,2865-28711, The data reported here suggest that although Arg-166 is important for activity it is not essential. The analysis of the kinetic data also suggests that th