Tyr306 near the C-terminus of protein phosphatase-1 affects enzyme stability and inhibitor binding

Previous deletion studies have suggested that Tyr306 has an important influence on the properties of protein phosphatase-1 (PP1). To test this inference, we constructed three site-directed mutants, PP1Y306A, PP1Y306K, and PP1Y306E. The specific activity of PP1Y306A was 3.5-fold higher than that of PP1wt, especially with K-R-Tp-I-R-R as substrate, and it also had a 13-fold higher Kcat and a 43-fold higher Kcat /Km . PP1Y306K and PP1Y306E, in that order, and were next in terms of increased activity. Use of the denaturant guanidine hydrochloride (GdnHCl) demonstrated that mutation of this site decreased enzyme stability. PP1Y306A and PP1Y306E lost all activity when incubated for 24 h in 0.6 M GdnHCl, and their fluorescence spectra confirmed the loss of stability. Because all three substitutions had a similar effect, we infer that the aromatic group of Tyr plays a crucial role in maintaining enzyme stability. Our results show that Tyr306 does affect the spatial conformation of the catalytic subunit of PP1 molecule. The IC50 of PP1Y306A for the inhibitor microcystin-LR was threefold higher than that of PP1wt, whereas those of PP1Y306E for tautomycin and norcantharidin were 15-fold and 10-fold higher, respectively. We conclude that Tyr306 plays an important role in enzyme stability and inhibitor binding.