Centro de Investigación en Alimentación y Desarrollo A.C., Hermosillo, 83304, Sonora, México.
Departamento de Ciencias Químico-Biológicas, Universidad de Sonora, Hermosillo, 83000, Sonora, México.
Mol Cell Biochem. 2021 Mar;476(3):1467-1475. doi: 10.1007/s11010-020-04010-3. Epub 2021 Jan 3.
The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the synthesis of glycine betaine (GB), an osmolyte and osmoprotectant. Also, it participates in several metabolic pathways in humans. All BADHs known have cysteine in the active site involved in the aldehyde binding, whereas the porcine kidney enzyme (pkBADH) also has a neighborhood cysteine, both sensitive to oxidation. The antineoplastic and immuno-suppressant pre-drug cyclophosphamide (CTX), and its bioactivation products, have two highly oxidating chlorine atoms. This work aimed to analyze the effect of CTX in the activity of porcine kidney betaine aldehyde dehydrogenase. PkBADH was incubated with varying CTX concentration (0 to 2.0 mM) at 25 °C and lost 50 % of its activity with 2.0 mM CTX. The presence of the coenzyme NAD (0.5 mM) decreased 95% the activity in 2.0 mM CTX. The substrate betaine aldehyde (0.05 and 0.4 mM, and the products NADH (0.1-0.5 mM) and GB (1 and 10 mM) did not have an effect on the enzyme inactivation by CTX. The reducing agents, dithiothreitol and β-mercaptoethanol, reverted the pkBADH inactivation, but reduced glutathione (GSH) was unable to restore the enzyme activity. Molecular docking showed that CTX could enter at the enzyme active site, where its chlorine atoms may interact with the catalytic and the neighboring cysteines. The results obtained show that CTX inactivates the pkBADH due to oxidation of the catalytic cysteine or because it oxidizes catalytic and neighborhood cysteine, forming a disulfide bridge with a concomitant decrease in the activity of the enzyme.
酶甜菜碱醛脱氢酶（BADH EC 1.2.1.8）催化甘氨酸甜菜碱（GB）的合成，GB 是一种渗透调节剂和渗透保护剂。此外，它还参与人体的几种代谢途径。所有已知的 BADH 在活性位点都含有半胱氨酸，参与醛的结合，而猪肾酶（pkBADH）也有一个邻近的半胱氨酸，两者都对氧化敏感。抗肿瘤和免疫抑制剂前药环磷酰胺（CTX）及其生物活化产物有两个高度氧化的氯原子。本工作旨在分析 CTX 对猪肾甜菜碱醛脱氢酶活性的影响。在 25°C 下，pkBADH 与不同浓度的 CTX（0 至 2.0 mM）孵育，当 CTX 浓度为 2.0 mM 时，酶活性丧失 50%。辅酶 NAD（0.5 mM）的存在使 2.0 mM CTX 中 95%的酶活性降低。底物甜菜碱醛（0.05 和 0.4 mM）和产物 NADH（0.1-0.5 mM）和 GB（1 和 10 mM）的存在对 CTX 引起的酶失活没有影响。还原剂二硫苏糖醇和β-巯基乙醇可使 pkBADH 失活逆转，但还原型谷胱甘肽（GSH）不能恢复酶活性。分子对接表明，CTX 可以进入酶的活性位点，其氯原子可能与催化和邻近的半胱氨酸相互作用。所得结果表明，CTX 通过氧化催化半胱氨酸或通过氧化催化和邻近半胱氨酸形成二硫键，同时降低酶活性，从而使 pkBADH 失活。
