Ma Ruixin, Kanders Erik, Sundh Ulla Beckman, Geng Meiyu, Ek Pia, Zetterqvist Orjan, Li Jin-Ping
Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden.
Biochem Biophys Res Commun. 2005 Nov 25;337(3):887-91. doi: 10.1016/j.bbrc.2005.09.134. Epub 2005 Sep 30.
Although protein histidine phosphorylation is estimated to account for about 6% of total protein phosphorylation in eukaryotes, knowledge on histidine phosphorylation and dephosphorylation is still limited. Recently, a few reports have appeared on a mammalian 14-kDa phosphohistidine phosphatase, also named protein histidine phosphatase. Molecular cloning of the protein has opened possibilities for exploring its properties and physiological role. In the present work, we have searched for potential active site residues in the human phosphohistidine phosphatase by point mutations of conserved histidine and arginine residues to alanine. When assayed by the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide, mutants H53A and H102A showed no detectable activity. Compared to the wild-type recombinant enzyme, the specific activity of mutant R45A was decreased by one order of magnitude, that of mutant R78A was decreased by about 30%, while that of mutant H81A was essentially unchanged. These results will facilitate future studies of the reaction mechanism, substrate binding, and molecular structure of the phosphohistidine phosphatase.
尽管在真核生物中，蛋白质组氨酸磷酸化估计约占总蛋白质磷酸化的6%，但关于组氨酸磷酸化和去磷酸化的知识仍然有限。最近，有几篇关于一种哺乳动物14 kDa磷酸组氨酸磷酸酶（也称为蛋白质组氨酸磷酸酶）的报道。该蛋白质的分子克隆为探索其性质和生理作用开辟了可能性。在本研究中，我们通过将保守的组氨酸和精氨酸残基突变为丙氨酸，在人磷酸组氨酸磷酸酶中寻找潜在的活性位点残基。当用含磷酸组氨酸的肽琥珀酰 - 丙氨酸 - 组氨酸（磷酸化） - 脯氨酸 - 苯丙氨酸 - 对硝基苯胺进行检测时，突变体H53A和H102A没有检测到活性。与野生型重组酶相比，突变体R45A的比活性降低了一个数量级，突变体R78A的比活性降低了约30%，而突变体H81A的比活性基本不变。这些结果将有助于未来对磷酸组氨酸磷酸酶的反应机制、底物结合和分子结构的研究。
