Encalada Rusely, Rojo-Domínguez Arturo, Rodríguez-Zavala José S, Pardo Juan P, Quezada Héctor, Moreno-Sánchez Rafael, Saavedra Emma
Departamento de Bioquímica, Instituto Nacional de Cardiología, Tlalpan, México DF, México.
FEBS J. 2009 Apr;276(7):2037-47. doi: 10.1111/j.1742-4658.2009.06939.x.
Phosphoglycerate kinase (EC 2.7.2.3) catalyzes reversible phosphoryl transfer from 1,3-bisphosphoglycerate to ADP to synthesize 3-phosphoglycerate and ATP during glycolysis. Phosphoglycerate kinases from several sources can use GDP/GTP as alternative substrates to ADP/ATP; however, the maximal velocities (V(m)) reached with the guanine nucleotides are approximately 50% of those displayed with the adenine nucleotides. By contrast, Entamoeba histolytica phosphoglycerate kinase (EC 2.7.2.10) is the only reported phosphoglycerate kinase displaying higher activity with GDP/GTP and lower affinities for the adenine nucleotides. To elucidate the molecular basis of the Entamoeba histolytica phosphoglycerate kinase selectivity for GDP/GTP, a conformational analysis was carried out on a homology model based on crystallographic structures of yeast and pig phosphoglycerate kinases. Some amino acid residues involved in the purine ring binding site not previously described were detected. Accordingly, Y239, E309 and V311 were replaced by site-directed mutagenesis in the Entamoeba histolytica phosphoglycerate kinase gene for the corresponding amino acid residues present in the adenine nucleotide-dependent phosphoglycerate kinases and the recombinant proteins were purified. Kinetic analysis of the enzymes showed that the single mutants Y239F, E309Q, E309M and V311L increased their catalytic efficiencies (V(m)/K(m)) with ADP/ATP as a result of both, increased V(m) and decreased K(m) values. Furthermore, a higher catalytic efficiency in the double mutant Y239F/E309M was achieved, which was mainly due to an increased affinity for ADP/ATP with a concomitant diminished affinity for GDP/GTP. The main Entamoeba histolytica phosphoglycerate kinase amino acid residues involved in the selectivity for guanine nucleotides were thus identified.
磷酸甘油酸激酶（EC 2.7.2.3）在糖酵解过程中催化1,3 - 二磷酸甘油酸向ADP的可逆磷酸转移，合成3 - 磷酸甘油酸和ATP。来自多种来源的磷酸甘油酸激酶可以使用GDP/GTP作为ADP/ATP的替代底物；然而，鸟嘌呤核苷酸达到的最大速度（V(m)）约为腺嘌呤核苷酸所显示速度的50%。相比之下，溶组织内阿米巴磷酸甘油酸激酶（EC 2.7.2.10）是唯一报道的对GDP/GTP具有更高活性且对腺嘌呤核苷酸亲和力较低的磷酸甘油酸激酶。为了阐明溶组织内阿米巴磷酸甘油酸激酶对GDP/GTP选择性的分子基础，基于酵母和猪磷酸甘油酸激酶的晶体结构对同源模型进行了构象分析。检测到一些以前未描述的参与嘌呤环结合位点的氨基酸残基。因此，在溶组织内阿米巴磷酸甘油酸激酶基因中，通过定点诱变将Y239、E309和V311替换为腺嘌呤核苷酸依赖性磷酸甘油酸激酶中存在的相应氨基酸残基，并纯化了重组蛋白。对这些酶的动力学分析表明，单突变体Y239F、E309Q、E309M和V311L由于V(m)增加和K(m)值降低，提高了以ADP/ATP为底物时的催化效率（V(m)/K(m)）。此外，双突变体Y239F/E309M实现了更高的催化效率，这主要是由于对ADP/ATP的亲和力增加，同时对GDP/GTP的亲和力降低。由此确定了溶组织内阿米巴磷酸甘油酸激酶中参与鸟嘌呤核苷酸选择性的主要氨基酸残基。
