Mutational analysis of a Ser/Thr phosphatase. Identification of residues important in phosphoesterase substrate binding and catalysis.

The Ser/Thr phosphoprotein phosphatases (PPases) display similarities in amino acid sequence and biochemical properties. Most members of this family require transition metal ions for activity. The smallest family member, the bacteriophage lambda PPase (lambda-PPase), has been successfully overexpressed in Escherichia coli, purified, and characterized (Zhuo, S., Clemens, J.C., Hakes, D.J., Barford, D., and Dixon, J. E. (1993) J. Biol. Chem. 268, 17754-17761). Site-directed mutagenesis has now been employed to define amino acid residues in lambda-PPase required for metal ion binding and catalysis. Conservative amino acid substitutions at residues Asp20, His22, Asp49, His76, and Glu77 affected lambda-PPase catalysis and metal ion binding, whereas substitutions at residues Arg53 and Arg73 affected catalysis and substrate binding. Each of these residues is invariant in all phosphoprotein phosphatases, suggesting that these residues may play important roles in binding and catalysis in all of the PPases. Computer-assisted sequence alignment further revealed that lambda-PPase residues Asp20, His22, Asp49, His76, Arg53, and Arg73 lie within three larger regions of PPase sequence identity with the consensus sequence (DXH-(approximately 25)-GDXXD-(approximately 25)-GNHD/E). This motif can be found in a wide variety of phosphoesterases unrelated to the PPases and defines structural and catalytic features utilized by a diverse group of enzymes for the hydrolysis of phosphate esters.