Gomez M S, Piper R C, Hunsaker L A, Royer R E, Deck L M, Makler M T, Vander Jagt D L
Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Mol Biochem Parasitol. 1997 Dec 1;90(1):235-46. doi: 10.1016/s0166-6851(97)00140-0.
Lactate dehydrogenase from the malarial parasite Plasmodium falciparum has many amino acid residues that are unique compared to any other known lactate dehydrogenase. This includes residues that define the substrate and cofactor binding sites. Nevertheless, parasite lactate dehydrogenase exhibits high specificity for pyruvic acid, even more restricted than the specificity of human lactate dehydrogenases M4 and H4. Parasite lactate dehydrogenase exhibits high catalytic efficiency in the reduction of pyruvate, kcat/Km = 9.0 x 10(8) min(-1) M(-1). Parasite lactate dehydrogenase also exhibits similar cofactor specificity to the human isoforms in the oxidation of L-lactate with NAD+ and with a series of NAD+ analogs, suggesting a similar cofactor binding environment in spite of the numerous amino acid differences. Parasite lactate dehydrogenase exhibits an enhanced kcat with the analog 3-acetylpyridine adenine dinucleotide (APAD+) whereas the human isoforms exhibit a lower kcat. This differential response to APAD+ provides the kinetic basis for the enzyme-based detection of malarial parasites. A series of inhibitors structurally related to the natural product gossypol were shown to be competitive inhibitors of the binding of NADH. Slight changes in structure produced marked changes in selectivity of inhibition of lactate dehydrogenase. 7-p-Trifluoromethylbenzyl-8-deoxyhemigossylic acid inhibited parasite lactate dehydrogenase, Ki = 0.2 microM, which was 65- and 400-fold tighter binding compared to the M4 and H4 isoforms of human lactate dehydrogenase. The results suggest that the cofactor site of parasite lactate dehydrogenase may be a potential target for structure-based drug design.
恶性疟原虫的乳酸脱氢酶具有许多与其他已知乳酸脱氢酶相比独特的氨基酸残基。这包括定义底物和辅因子结合位点的残基。然而，寄生虫乳酸脱氢酶对丙酮酸表现出高度特异性，甚至比人类乳酸脱氢酶M4和H4的特异性更受限制。寄生虫乳酸脱氢酶在丙酮酸还原反应中表现出高催化效率，kcat/Km = 9.0 x 10(8) min(-1) M(-1)。在L-乳酸与NAD+以及一系列NAD+类似物的氧化反应中，寄生虫乳酸脱氢酶也表现出与人类同工酶相似的辅因子特异性，这表明尽管存在众多氨基酸差异，但辅因子结合环境相似。寄生虫乳酸脱氢酶与类似物3-乙酰吡啶腺嘌呤二核苷酸（APAD+）反应时kcat增强，而人类同工酶的kcat较低。这种对APAD+的差异反应为基于酶的疟原虫检测提供了动力学基础。一系列与天然产物棉酚结构相关的抑制剂被证明是NADH结合的竞争性抑制剂。结构上的微小变化导致乳酸脱氢酶抑制选择性的显著变化。7-p-三氟甲基苄基-8-脱氧半棉酚酸抑制寄生虫乳酸脱氢酶，Ki = 0.2 microM，与人类乳酸脱氢酶的M4和H4同工酶相比，其结合紧密程度分别高65倍和400倍。结果表明，寄生虫乳酸脱氢酶的辅因子位点可能是基于结构的药物设计的潜在靶点。
