Identification and characterization of the core region of protein phosphatase-1 |
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Authors: | Bai J. Wang Wei Tang Peng Zhang Qun Wei |
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Affiliation: | 1.Department of Biochemistry and Molecular Biology,Beijing Normal University, Beijing Key Lab.,Beijing,People’s Republic of China |
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Abstract: | 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. |
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