Kavanagh Kathryn L, Elling Robert A, Wilson David K
Section of Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
Biochemistry. 2004 Feb 3;43(4):879-89. doi: 10.1021/bi035108g.
While within a human host the opportunistic pathogen Toxoplasma gondii relies heavily on glycolysis for its energy needs. Lactate dehydrogenase (LDH), the terminal enzyme in anaerobic glycolysis necessary for NAD(+) regeneration, therefore represents an attractive therapeutic target. The tachyzoite stage lactate dehydrogenase (LDH1) from the parasite T. gondii has been crystallized in apo form and in ternary complexes containing NAD(+) or the NAD(+)-analogue 3-acetylpyridine adenine dinucleotide (APAD(+)) and sulfate or the inhibitor oxalate. Comparison of the apo and ternary models shows an active-site loop that becomes ordered upon substrate binding. This active-site loop is five residues longer than in most LDHs and necessarily adopts a different conformation. While loop isomerization is fully rate-limiting in prototypical LDHs, kinetic data suggest that LDH1's rate is limited by chemical steps. The importance of charge neutralization in ligand binding is supported by the complexes that have been crystallized as well as fluorescence quenching experiments performed with ligands at low and high pH. A methionine that replaces a serine residue and displaces an ordered water molecule often seen in LDH structures provides a structural explanation for reduced substrate inhibition. Superimposition of LDH1 with human muscle- and heart-specific LDH isoforms reveals differences in residues that line the active site that increase LDH1's hydrophobicity. These differences will aid in designing inhibitors specific for LDH1 that may be useful in treating toxoplasmic encephalitis and other complications that arise in immune-compromised individuals.
在人类宿主体内，机会性致病原弓形虫在很大程度上依赖糖酵解来满足其能量需求。乳酸脱氢酶（LDH）是无氧糖酵解中NAD(+)再生所必需的末端酶，因此是一个有吸引力的治疗靶点。来自弓形虫的速殖子阶段乳酸脱氢酶（LDH1）已以无配体形式以及包含NAD(+)或NAD(+)类似物3-乙酰吡啶腺嘌呤二核苷酸（APAD(+)）和硫酸盐或抑制剂草酸盐的三元复合物形式结晶。无配体模型和三元模型的比较显示，一个活性位点环在底物结合时变得有序。这个活性位点环比大多数LDH中的长五个残基，并且必然采用不同的构象。虽然在典型的LDH中，环异构化是完全限速的，但动力学数据表明LDH1的速率受化学步骤限制。已结晶的复合物以及在低pH和高pH下用配体进行的荧光猝灭实验支持了电荷中和在配体结合中的重要性。一个取代丝氨酸残基并取代LDH结构中常见的有序水分子的甲硫氨酸为减少底物抑制提供了结构解释。将LDH1与人类肌肉和心脏特异性LDH同工型进行叠加显示，活性位点内衬的残基存在差异，这增加了LDH1的疏水性。这些差异将有助于设计对LDH1具有特异性的抑制剂，这些抑制剂可能有助于治疗弓形虫性脑炎以及免疫功能低下个体出现的其他并发症。
