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 thi s family require transition metal ions for activity. The smallest fami ly member, the bacteriophage lambda PPase (lambda-PPase), has been suc cessfully overexpressed in Escherichia coli, purified, and characteriz ed (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 r equired for metal ion binding and catalysis. Conservative amino acid s ubstitutions at residues Asp(20), His(22), ASp(49), His(76), and Glu(7 7) affected lambda-PPase catalysis and metal ion binding, whereas subs titutions at residues Arg(53) and Arg(73) affected catalysis and subst rate binding. Each of these residues is invariant in all phosphoprotei n phosphatases, suggesting that these residues may play important role s in binding and catalysis in all of the PPases. Computer-assisted seq uence alignment further revealed that lambda-PPase residues Asp(20), H is(22), Asp(49), His(76), Arg(53), and Arg(73) lie within three larger regions of PPase sequence identity with the consensus sequence (DXH-( similar to 25)-GDXXD-(similar to 25)-GNHD/E). This motif can be found in a wide variety of phosphoesterases unrelated to the PPases and defi nes structural and catalytic features utilized by a diverse group of e nzymes for the hydrolysis of phosphate esters.