Comparative structural analysis and kinetic properties of lactate dehydrogenases from the four species of human malarial parasites
Comparative structural analysis and kinetic properties of lactate dehydrogenases from the four species of human malarial parasites
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DOI：
10.1021/bi049892w
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发表时间：
2004-05-25
期刊：
影响因子：
2.9
通讯作者：
Jagt, DLV
中科院分区：
文献类型：
--
作者：
Brown, WM;Yowell, CA;Jagt, DLV
寄生虫乳酸脱氢酶（pLDH）是一个潜在的药物靶点，新的抗疟药，由于寄生虫依赖糖酵解ATP的生产。从所有四种人疟疾寄生虫的pLDH克隆，表达，并分析可能被开发用于药物开发的结构和动力学特性。来自间日疟原虫、三日疟原虫和卵形疟原虫的pLDH与来自恶性疟原虫的pLDH具有90-92%的同一性。催化剂残留物相同。在大多数LDH中保守的残基1250和T246在所有pLDH中被脯氨酸取代。pLDH在底物特异性环中含有相同的五个氨基酸插入物（DKEWN）。在辅因子位点内，来自恶性疟原虫和三日疟原虫的pLDH是相同的，而来自间日疟原虫和卵形疟原虫的pLDH有一个取代。来自间日疟原虫、卵形疟原虫和三日疟原虫的pLDH与来自恶性疟原虫的pLDH的晶体结构的同源性建模得到几乎相同的结构。然而，动力学特性和针对辅因子结合位点的抑制剂的敏感性显著不同。丙酮酸和乳酸的米氏常数相差8-9倍; NADH、NAD（+）和NAD（+）类似物3-乙酰基吡啶腺嘌呤二核苷酸的米氏常数相差高达4倍。抑制剂的解离常数相差高达21倍。所有四个pLDH的辅因子位点的抑制剂的结合的分子对接研究预测类似的取向，与对接的配体定位在辅因子位点的烟酰胺末端。pH研究表明，在pH 6-8范围内，抑制剂结合与pH无关，这表明特定抑制剂的解离常数差异不是由于四种pLDH中活性位点pK值的改变。
Parasite lactate dehydrogenase (pLDH) is a potential drug target for new antimalarials owing to parasite dependence on glycolysis for ATP production. The pLDH from all four species of human malarial parasites were cloned, expressed, and analyzed for structural and kinetic properties that might be exploited for drug development. pLDH from Plasmodium vivax, malariae, and ovale exhibit 90-92% identity to pLDH from Plasmodium falciparum. Catalytic residues are identical. Resides 1250 and T246, conserved in most LDH, are replaced by proline in all pLDH. The pLDH contain the same five-amino acid insert (DKEWN) in the substrate specificity loops. Within the cofactor site, pLDH from P. falciparum and P. malariae are identical, while pLDH from P. vivax and P. ovale have one substitution. Homology modeling of pLDH from P. vivax, ovale, and malariae with the crystal structure of pLDH from P. falciparum gave nearly identical structures. Nevertheless, the kinetic properties and sensitivities to inhibitors targeted to the cofactor binding site differ significantly. Michaelis constants for pyruvate and lactate differ 8-9-fold; Michaelis constants for NADH, NAD(+), and the NAD(+) analogue 3-acetylpyridine adenine dinucleotide differ up to 4-fold. Dissociation constants for the inhibitors differ up to 21-fold. Molecular docking studies of the binding of the inhibitors to the cofactor sites of all four pLDH predict similar orientations, with the docked ligands positioned at the nicotinamide end of the cofactor site. pH studies indicate that inhibitor binding is independent of pH in the pH 6-8 range, suggesting that differences in dissociation constants for a specific inhibitor are not due to altered active site pK values among the four pLDH.