Lillo C, Ruoff P
Rogaland University Center, Stavanger, Norway.
J Biol Chem. 1992 Jul 5;267(19):13456-9.
In the absence of NADH, at 25 degrees C, partially purified NADH:nitrate reductase undergoes an approximately 50% reduction of its initial activity during 2 h. With the increase of inactivation, the NADH and nitrite concentration time curves become typical "sigmoidal," i.e. the reaction velocity of the nitrate reductase catalyzed reaction goes through a maximum before equilibrium is reached. About 80% of the original activity of nitrate reductase is restored when the enzyme is incubated for 2 min with 200 microM NADH or NADPH. Also other NADH substrate analogues have similar effects in restoring the lost activity. After incubation with the reduced pyridine nucleotides, the sigmoidal appearance of the NADH concentration time curve disappears almost completely. Despite the fact that NADPH increases the activity of the enzyme, NADPH does not show any competition with the NADH-binding site of nitrate reductase and does not produce nitrite in the absence of NADH. It is therefore concluded that there must be an additional allosteric site which binds either NADH or NADPH, or other pyridine nucleotides with the effect of increasing the activity of the enzyme. A kinetic model is presented which simulates the observed experimental findings.
在没有NADH的情况下，25摄氏度时，部分纯化的NADH：硝酸还原酶在2小时内其初始活性会降低约50%。随着失活程度的增加，NADH和亚硝酸盐浓度-时间曲线变得典型的“s形”，即硝酸还原酶催化反应的反应速度在达到平衡之前会经历一个最大值。当酶与200微摩尔的NADH或NADPH孵育2分钟时，约80%的硝酸还原酶原始活性得以恢复。其他NADH底物类似物在恢复丧失的活性方面也有类似效果。与还原型吡啶核苷酸孵育后，NADH浓度-时间曲线的s形几乎完全消失。尽管NADPH会增加酶的活性，但NADPH与硝酸还原酶的NADH结合位点没有竞争，且在没有NADH的情况下不会产生亚硝酸盐。因此得出结论，必然存在一个额外的变构位点，它能结合NADH或NADPH，或其他吡啶核苷酸，从而增加酶的活性。本文提出了一个动力学模型，该模型模拟了观察到的实验结果。
