Mutational Analysis of Substrate Recognition by Protein Phosphatase 1

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Abstract
The role of residues that are involved in substrate recognition by rabbit muscle protein phosphatase 1α (PP1) was investigated by site-directed mutagenesis and kinetic analyses using phosphorylase a, RII peptide, Kemptide, and p-nitrophenyl phosphate as substrates. The atomic structure of PP1 has shown the active site to be at the confluence of three shallow grooves, a C-terminal groove, an acidic groove, and a hydrophobic groove. Mutations of residues D208, D210, D212, E218, D220, E252, D253, E256, E275, and D277 in the acidic groove, of R221, W206, and Y134, which have been suggested to be involved in substrate binding, and of residues C127, I130, and D197 in the hydrophobic groove were examined. Our results show that mutations in the acidic groove lead to modest changes in substrate binding, consistent with a role of the acidic residues in forming a negatively charged surface well for binding of peptides with basic N-termini. Severe effects on Vmax were observed for mutants of R221, D208, and W206. These results are consistent with the proposal that the R221 plays an important role as a phosphate oxygen ligand that positions the substrate for catalysis. The kinetic behavior of mutants at W206 and D208 can be explained by the observation that, together with R221, these residues form the microenvironment which dictates the orientation of the imidazole ring of H248, one of the metal binding ligands, as well as contributing to the orientation of R221 itself.