THE MOLECULAR-BASIS OF THE DIFFERING KINETIC-BEHAVIOR OF THE 2 LOW-MOLECULAR-MASS PHOSPHOTYROSINE PROTEIN PHOSPHATASE ISOFORMS

The low molecular mass phosphotyrosine protein phosphatase is a cytoso lic enzyme of 18 kDa, Mammalian species contain a single gene that cod ifies for two distinct isoenzymes; they are produced through alternati ve splicing and thus differ only in the sequence from residue 40 to re sidue 73, Isoenzymes differ also in substrate specificity and in the s ensitivity to activity modulators, In our study, we mutated a number o f residues included in the alternative 40-73 sequence by substituting the residues present in the type 2 isoenzyme with those present in typ e 1 and subsequently examined the kinetic properties of the purified m utated proteins, The results enabled us to identify the molecular site that determines the kinetic characteristics of each isoform; the resi due in position 50 plays the main role in the determination of substra te specificity, while the residues in both positions 49 and 50 are inv olved in the strong activation of the type 2 low M(r) phosphotyrosine protein phosphatase isoenzyme by purine compounds such as guanosine an d cGMP, The sequence 49-50 is included in a loop whose N terminus is l inked to the beta 2-strand and whose C terminus is linked to the alpha 2-helix; this loop is very near the active site pocket, Our findings suggest that this loop is involved both in the regulation of the enzym e activity and in the determination of the substrate specificity of th e two low M(r) phosphotyrosine protein phosphatase isoenzymes.