MUTATIONAL ANALYSIS OF SUBSTRATE RECOGNITION BY PROTEIN PHOSPHATASE-1

The role of residues that are involved in substrate recognition by rab bit muscle protein phosphatase 1 alpha (PP1) was investigated by site- directed mutagenesis and kinetic analyses using phosphorylase a, RII p eptide, Kemptide, and p-nitrophenyl phosphate as substrates. The atomi c structure of PP1 has shown the active site to be at the confluence o f 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 bind ing, and of residues C127, I130, and D197 in the hydrophobic groove we re 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 bind ing of peptides with basic N-termini. Severe effects on V-max were obs erved for mutants of R221, D208, and W206. These results are consisten t with the proposal that the R221 plays an important role as a phospha te oxygen ligand that positions the substrate for catalysis. The kinet ic behavior of mutants at W206 and D208 can be explained by the observ ation that, together with R221, these residues form the microenvironme nt which dictates the orientation of the imidazole ring of H248, one o f the metal binding ligands, as well as contributing to the orientatio n of R221 itself.