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

阿平添加于 2011-6-24 21:07 | 1560 次阅读 | 0 个评论

作者
Wang BJ, Tang W, Zhang P, Wei Q
摘要
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 K(cat) and a 43-fold higher K(cat) /K(m) . 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 IC(50) 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) 2011 IUBMB IUBMB Life, 63(7): 574-581, 2011.
详细资料
- 文献种类: Journal Article
- 期刊名称: IUBMB Life
- 期刊缩写: IUBMB Life
- 期卷页: 2011年第63卷第7期 574-581页
- 地址: Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory, Beijing, People's Republic of China
- ISBN: 1521-6543
- 备注:PMID:21698763
学科领域生物医药 » 生物学
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PP1
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