Zhang J, Zhang Z, Brew K, Lee E Y
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Biochemistry. 1996 May 21;35(20):6276-82. doi: 10.1021/bi952954l.
A mutational analysis of rabbit skeletal muscle protein phosphatase-1 was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli. The selection of the sites to be mutated was based on sequence alignments which showed the existence of a number of invariant residues when eukaroytic Ser/Thr protein phosphatases were compared with bacteriophage phosphatases and adenosinetetraphosphatase [Barton et al. (1995) Eur. J. Biochem. 220, 225-237]. In other studies, it had been shown that PP1 is a metalloprotein [Chu et al. (1996) J. Biol. Chem. 271, 2574-2577], and in this study, we have largely focused on invariant histidine and aspartate residues which may be involved in metal binding. The residues which were mutated were H66, H125, H173, H248, D64, D71, D92, D95, N124, and R96E. The results showed that mutation of H66, H248, D64, and D92 resulted in severe loss of catalytic function. Mutation of D95, N124, and R96 also led to loss of function, while attempts to mutate H125 and H173 led to production of insoluble, inactive proteins. The results of the mutational analysis are consistent with the involvement of conserved His and Asp residues in metal binding, and are discussed in the context of the recently described crystal structure of PP1 [Goldberg et al. (1995) Nature, 376, 745-753], which reveals that PP1 possesses a bimetallic center at the active site. The behavior of the D95, R96, and N124 mutants supports a catalytic mechanism involving nucleophilic attack by a hydroxide ion with H125 functioning as a proton donor to the leaving alcohol group.
通过对在大肠杆菌中表达的重组蛋白进行定点诱变，对兔骨骼肌蛋白磷酸酶-1进行了突变分析。待突变位点的选择基于序列比对，该比对显示，当将真核丝氨酸/苏氨酸蛋白磷酸酶与噬菌体磷酸酶和腺苷四磷酸酶进行比较时，存在许多不变残基[巴顿等人（1995年），《欧洲生物化学杂志》220卷，225 - 237页]。在其他研究中，已表明PP1是一种金属蛋白[朱等人（1996年），《生物化学杂志》271卷，2574 - 2577页]，在本研究中，我们主要关注可能参与金属结合的不变组氨酸和天冬氨酸残基。被突变的残基有H66、H125、H173、H248、D64、D71、D92、D95、N124以及R96E。结果表明，H66、H248、D64和D92的突变导致催化功能严重丧失。D95、N124和R96的突变也导致功能丧失，而对H125和H173进行突变的尝试导致产生不溶性的无活性蛋白。突变分析结果与保守的组氨酸和天冬氨酸残基参与金属结合一致，并在最近描述的PP1晶体结构[戈德堡等人（1995年），《自然》376卷，745 - 753页]的背景下进行了讨论，该晶体结构揭示PP1在活性位点拥有一个双金属中心。D95、R96和N124突变体的行为支持一种催化机制，即涉及氢氧根离子的亲核攻击，其中H125作为离去醇基团的质子供体。
