BACKGROUND Phosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn2+ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis. METHODS Key residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography. RESULTS Mutation of residues D269 and H232, which comprise part of the coordination sphere of Mn2+, reduced kcat by 14-fold, and significantly increased the Km values for Mn2+ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the Km value for MgATP and reduced kcat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the Km value for OAA and reduced kcat respectively. CONCLUSIONS Our results show that mutation of residues involved in coordinating OAA, MgATP and Mn2+ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK. GENERAL SIGNIFICANCE In higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.

中文翻译：

---

大肠杆菌磷酸烯醇丙酮酸羧激酶突变体的动力学和结构分析。

背景技术磷酸烯醇丙酮酸羧激酶（PEPCK）是糖异生途径中的代谢酶，其催化草酰乙酸酯（OAA）可逆转化为磷酸烯醇丙酮酸（PEP）和CO 2。大肠杆菌PEPCK的底物是OAA和MgATP，其中Mn2 +作为辅助因子。对PEPCK结构的分析表明，催化过程中涉及底物/辅因子配位的氨基酸残基。方法突变与大肠杆菌底物结合的关键底物和辅因子。纯化的突变酶用于动力学测定。使用X射线晶体学确定某些突变酶的结构。结果残基D269和H232突变，构成Mn2 +配位域的一部分，使kcat降低14倍，并显着提高了Mn2 +和OAA的Km值。K254突变是P环基序中与MgATP相互作用的关键残基，可显着提高MgATP的Km值并降低kcat。R65和R333是与OAA相互作用的关键残基。R65Q和R333Q突变分别显着提高了OAA的Km值和降低的kcat。结论我们的结果表明，参与协调OAA，MgATP和Mn2 +的残基突变显着降低了PEPCK活性。K254在磷酰基转移中起重要作用，而R333参与OAA脱羧和大肠杆菌PEPCK的磷酰基转移。一般意义在包括人类在内的高等生物中，PEPCK有助于调节血糖水平，因此PEPCK是非胰岛素依赖型糖尿病患者的潜在药物靶标。