Crystal structure and catalytic activity of the PPM1K N94K mutant
Crystal structure and catalytic activity of the PPM1K N94K mutant
复制标题
PPM1K N94K突变体的晶体结构和催化活性
DOI：
10.1111/jnc.14631
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发表时间：
2019-02-01
影响因子：
4.7
通讯作者：
Zhang, Peng-ju
中科院分区：
文献类型：
--
作者：
Dolatabad, Meisam Rostaminasab;Guo, Lu-lu;Zhang, Peng-ju
蛋白磷酸酶Mg 2 +/Mn 2+依赖性1 K（PPM 1 K），也称为PP 2Cm或支链α-酮酸脱氢酶复合磷酸酶，是金属依赖性磷酸酶家族的成员，也是一种重要的代谢调节剂。PPM 1 K中的单核苷酸多态性（SNP）导致蛋白质功能缺陷，已被发现与许多人类疾病相关，如心血管疾病、枫糖浆尿病、2型糖尿病和神经系统疾病。PPM 1 K N94 K是由人PPM 1 K编码序列中的一个SNP产生的经鉴定的错义突变体。然而，N94 K突变体对其活性和结构性质的影响尚未确定。在这里，我们进行了详细的酶学研究，在pNPP或磷酸肽底物和晶体学分析的野生型和N94 K PPM 1 K的存在下，使用稳态动力学。PPM 1 K-N94 K突变显著降低了其Mg 2+依赖的催化活性，结构分析表明N94 K突变导致活性位点中与Mg 2+配位的关键残基发生构象变化。具体地，三个Mg 2+位于PPM 1 K N94 K的活性位点，而不是PPM 1 K野生型中的两个Mg 2+。因此，我们的研究结果为金属离子依赖的PPM 1 K-N94 K磷酸酶活性提供了结构基础。
Protein Phosphatase Mg2+/Mn2+-Dependent 1K (PPM1K),also named as PP2Cm or branched-chain alpha-ketoacid dehydrogenase complex phosphatase, is a member of the metal-dependent phosphatase family and an important metabolic regulator. Single nucleotide polymorphisms (SNPs) in PPM1K contributing to protein functional defects have been found to be associated with numerous human diseases, such as cardiovascular disease, maple syrup urine disease, type 2 diabetes, and neurological disease. PPM1K N94K is an identified missense mutant produced by one of the SNPs in the human PPM1K coding sequence. However, the effects of the N94K mutant on its activity and structural property have not been defined. Here, we performed a detailed enzymological study using steady-state kinetics in the presence of pNPP or phospho-peptide substrates and crystallographic analyses of the wild-type and N94K PPM1K. The PPM1K-N94K significantly impaired its Mg2+-dependent catalytic activity and structural analysis demonstrated that the N94K mutation induced a conformational change in the key residue in coordinating the Mg2+ in the active site. Specifically, three Mg2+ were located in the active site of the PPM1K N94K instead of two Mg2+ in the PPM1K wild type. Therefore, our results provide a structure basis for the metal ion-dependent PPM1K-N94K phosphatase activity.