Coordinación de Ciencia de los Alimentos, Centro de Investigación en Alimentación y Desarrollo AC, Sonora, Mexico.
Chem Biol Interact. 2011 May 30;191(1-3):159-64. doi: 10.1016/j.cbi.2011.01.030. Epub 2011 Feb 4.
Concentrated urine formation in the kidney is accompanied by conditions that favor the accumulation of reactive oxygen species (ROS). Under hyperosmotic conditions, medulla cells accumulate glycine betaine, which is an osmolyte synthesized by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). All BADHs identified to date have a highly reactive cysteine residue at the active site, and this cysteine is susceptible to oxidation by hydrogen peroxide. Porcine kidney BADH incubated with H(2)O(2) (0-500 μM) lost 25% of its activity. However, pkBADH inactivation by hydrogen peroxide was limited, even after 120 min of incubation. The presence of coenzyme NAD(+) (10-50 μM) increased the extent of inactivation (60%) at 120 min of reaction, but the ligands betaine aldehyde (50 and 500 μM) and glycine betaine (100 mM) did not change the rate or extent of inactivation as compared to the reaction without ligand. 2-Mercaptoethanol and dithiothreitol, but not reduced glutathione, were able to restore enzyme activity. Mass spectrometry analysis of hydrogen peroxide inactivated BADH revealed oxidation of M278, M243, M241 and H335 in the absence and oxidation of M94, M327 and M278 in the presence of NAD(+). Molecular modeling of BADH revealed that the oxidized methionine and histidine residues are near the NAD(+) binding site. In the presence of the coenzyme, these oxidized residues are proximal to the betaine aldehyde binding site. None of the oxidized amino acid residues participates directly in catalysis. We suggest that pkBADH inactivation by hydrogen peroxide occurs via disulfide bond formation between vicinal catalytic cysteines (C288 and C289).
肾脏中的浓缩尿液形成伴随着有利于活性氧物质 (ROS) 积累的条件。在高渗条件下，髓质细胞积累甘氨酸甜菜碱，这是甜菜碱醛脱氢酶 (BADH，EC 1.2.1.8) 合成的一种渗透剂。迄今为止鉴定的所有 BADH 在活性部位都具有高度反应性的半胱氨酸残基，并且该半胱氨酸易被过氧化氢氧化。与 H(2)O(2) (0-500 μM) 孵育的猪肾 BADH 失去了 25%的活性。然而，即使孵育 120 分钟，过氧化物酶对猪肾 BADH 的失活也是有限的。辅酶 NAD(+) (10-50 μM) 的存在增加了 120 分钟反应时的失活程度（60%），但配体甜菜碱醛（50 和 500 μM）和甘氨酸甜菜碱（100 mM）与无配体的反应相比，没有改变失活的速率或程度。2-巯基乙醇和二硫苏糖醇，但不是还原型谷胱甘肽，能够恢复酶活性。对过氧化物酶失活的 BADH 进行质谱分析表明，在没有 NAD(+) 的情况下，M278、M243、M241 和 H335 发生氧化，在有 NAD(+) 的情况下，M94、M327 和 M278 发生氧化。BADH 的分子建模表明，氧化的蛋氨酸和组氨酸残基靠近 NAD(+) 结合位点。在辅酶存在的情况下，这些氧化的残基靠近甜菜碱醛结合位点。没有一个氧化的氨基酸残基直接参与催化。我们认为，过氧化物酶对猪肾 BADH 的失活是通过相邻催化半胱氨酸 (C288 和 C289) 之间形成二硫键引起的。
