Schäfer H G, Jacobi T, Eichhorn H, Woenckhaus C
Biol Chem Hoppe Seyler. 1986 Sep;367(9):969-80. doi: 10.1515/bchm3.1986.367.2.969.
Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.
从6-氯嘌呤核糖核苷和NAD⁺出发，通过柔性间隔基将重氮芳基或芳香族亚氨酯基团引入辅酶的无功能腺嘌呤部分，得到了不同的NAD⁺反应类似物。这些类似物分别与脱氢酶的不同氨基酸残基反应，形成稳定的脒或偶氮桥。在辅酶、酶和假底物形成三元复合物后，反应性间隔基锚定在活性位点附近。因此，即使复合物分解后，辅酶仍与蛋白质共价结合。加入底物后，共价结合的辅酶转化为二氢形式。在酶促试验中，修饰后的脱氢酶显示出天然酶80 - 90%的比活性，但它们需要显著更高浓度的游离NAD⁺才能达到这些值。二氢辅酶可以被诸如硫酸吩嗪等氧化剂或第二个酶系统重新氧化。研究了各种辅酶再生系统；修饰的酶是猪心乳酸脱氢酶和马肝醇脱氢酶；辅助酶是酵母和肝脏的醇脱氢酶、猪心乳酸脱氢酶、谷氨酸脱氢酶和丙氨酸脱氢酶。心肌乳酸脱氢酶受丙酮酸抑制。以丙氨酸脱氢酶作为辅助酶，辅酶得以再生，反应产物丙酮酸被去除。该系统成功地将乳酸定量转化为L-丙氨酸。二元酶系统的热稳定性表明共价结合的辅酶与两种脱氢酶之间存在相互作用；两个结合位点似乎在竞争辅酶。对不同修饰程度的脱氢酶进行比较表明，产物形成主要取决于掺入辅酶的量。
