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.

C-terminal regions of the protein phosphatases PP1 and PP2B were seldom studied. C-terminal 24 amino acids of PP1 was deleted, its enzymatic activity increased 3-fold while its stability declined. When the truncated PP1 was fused with the terminal (residues 483-511) of PP2B, both its enzymatic activity and its stability remained low. This indicates that the termini of PP2B and PP1 have inhibitory effect on the catalytic domain of PP1. PP1-(1-306) and PP1wt differ in their activation by metal ions, showing that the sites interacting with metal ions are not located in its C-terminus; while metal ions activated notably to PP1/PP2B chimera. In addition, the sensitivity results of PP1-(1-306) to the inhibitors, TM and NCTD, proved that these two inhibitors also did not bind to the C-terminus. However, the IC(50)s of PP1/PP2B chimera were higher than for PP1-(1-306), indicating that the C-terminal region interferes interactions with these inhibitors to some extent. Although 483-511 segment of PP2B was not the functional domain, it played important role in interaction with metal ions and inhibitors. It further indicates although PP1 and PP2B have high sequence identity, their non-conserved termini have different roles.

PP1, PP2A and PP2B all belong to PPP family of serine/threonine protein phosphatases. Their primary structures are highly conserved, particularly in the catalytic domain. In order to obtain correlative information about this conserved region, we constructed N-, C-deletion and N/C double-deletion mutants. We found that the N-and Csingle-deletion mutants exhibited higher enzymatic activities, while specific activity of N/C double-deletion mutant PP1 (9-306) did not notably change. The results of kinetics analysis showed that kcat and kcat/Km increased about 16-fold in the single-deletion mutants; while the two parameters of the double-deletion were lower than the single-deletions. We further explored stability of all mutants in existing denaturant guanidine hydrochloride (GdnHCl). It was noticeable that stability of PP1-(9-306) in all mutants was the highest. We speculated that PP1-(9-306) maybe retains a compact spherical structure, thus accordingly affected molecular catalysis. On the other hand the structures of single-deletion mutants were relatively relaxed, which were able to bind substrate easily, so activities of single-deletion mutants were higher than that of double-deletion mutant. We therefore deduced that PP1-(9-306) may be close to core region of PP1 molecule. In order to further solidify this idea, we used fluorescence spectra method to explore changes of space conformation. We found that emission peaks of all single-deletions were blue shifted in different degree in the absence of denaturant, while emission peak of N/C double-deletion mutant did not change obviously compared with that of the wild-type PP1. Conformation change of N/C double-deletion mutant was significantly less than those of single-deletion mutants in different GdnHCl concentration.