Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Effects of pH, salts, temperature, and source of NADPH on enzyme activity and substrate specificity studies.
Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Effects of pH, salts, temperature, and source of NADPH on enzyme activity and substrate specificity studies.
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来自抗甲氨蝶呤干酪乳杆菌的二氢叶酸还原酶。
DOI：
10.1016/0003-9861(77)90263-6
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发表时间：
1977
影响因子：
3.9
通讯作者：
R. Dunlap
中科院分区：
文献类型：
--
作者：
T. Williams;T. K. Lee;R. Dunlap
利用商业来源的NADPH对嗜紫蝶呤抗性乳酸菌的纯二氢叶酸还原酶进行活性分析，得到了一系列非线性分析图，其形状既依赖于pH值，又让人想起经典的产物抑制。实验示踪的曲线程度取决于商业NADPH的来源和年龄，并随着pH从7.5降至5.0而增强。在这些条件下，发现了一个“伪”pH-活性谱，在pH 7.0和7.5之间，蛋白质的最大比活性为9单位/毫克。相比之下，新鲜制备的NADPH在pH为8.5至5.0的范围内提供了严格的线性分析跟踪，产生的比活性一致高于商用NADPH。新的pH-活性谱在pH 5.0 ~ 6.0之间具有广泛的最优特征，在不添加盐的情况下，0.1 m磷酸钾的最大特异性活性为24.9单位/ mg。用商用NADPH观察到的弯曲现象和伪ph最佳是由于辅酶制剂中存在少量但有效的抑制杂质。一价盐（~0.1m）和二价盐（~0.05m）的最佳浓度对酶的活性为1.5 ~ 1.7倍，最大比活性为34 ~ 39单位/mg。在0.8mTris-acetate缓冲液中，pH为5.5，也观察到类似程度的活化。单价盐浓度高于0.5ma，二价盐浓度高于0.2ma时，盐依赖性激活降低，在某些情况下，活性受到抑制。底物特异性研究表明，叶酸在饱和水平下的v值是二氢叶酸的1%。Deamino-NADPH产率比NADPH高1.4倍，acetylpyridine-NADPH和thio-NADPH产率分别比天然辅酶低6.5倍和235倍。凝胶电泳研究也反映了NADPH及其类似物相互作用形成稳定二元配合物的选择性趋势。NADPH、脱氨基-NADPH、硫代-NADPH和乙酰吡啶-NADPH形成稳定的酶与紫蝶呤三元配合物。虽然二氢叶酸和NADP+及其类似物都不能与l形成稳定的配合物。酪蛋白二氢叶酸还原酶，NADP+和脱氨基-NADP+与酶和二氢叶酸相互作用，生成稳定的三元配合物。
Activity analyses of pure dihydrofolate reductase from amethopterin-resistantLactobacillus caseiconducted with commercial sources of NADPH yielded a progression of nonlinear assay tracings whose shapes were both pH dependent and reminiscent of classical product inhibition. The extent of curving of the assay tracings was dependent on the source and age of the commercial NADPH and was enhanced as the pH was decreased from 7.5 to 5.0. Under these conditions a “pseudo”-pH-activity profile, exhibiting a maximal specific activity of 9 units/mg of protein between pH 7.0 and 7.5, was found. In contrast, freshly prepared NADPH provided strictly linear assay tracings over the pH range of 8.5 to 5.0, yielding uniformly higher specific activities than those observed with commercial NADPH. The new pH-activity profile was characterized by a broad optimum between pH 5.0 and 6.0, with a maximal specificity activity of 24.9 units/ mg in 0.1mpotassium phosphate in the absence of added salt. The curving phenomenon and pseudo-pH optimum observed with commercial NADPH is attributed to the presence of minor but potent inhibitory impurities in these coenzyme preparations. Optimal concentrations of monovalent (~0.1m) and divalent (~0.05m) salts activated the enzyme between 1.5- and 1.7-fold, resulting in maximal specific activities in the range of 34 to 39 units/mg. A similar extent of activation was observed in 0.8mTris-acetate buffer, pH 5.5. At concentrations of monovalent salts above 0.5mand of divalent salts above 0.2ma reduction in salt-dependent activation and, in some cases, inhibition of activity were obtained. Substrate specificity studies indicated that theVfor folate at saturating levels is 1% of that for dihydrofolate. Deamino-NADPH yieldedVvalues 1.4-fold higher than that for NADPH, while acetylpyridine-NADPH and thio-NADPH provided values 6.5- and 235-fold lower, respectively, than the value with the natural coenzyme. Gel electrophoresis studies reflected a similar trend of selectivity in the interaction of NADPH and its analogs to form stable binary complexes. Stable ternary complexes of enzyme and amethopterin were formed with NADPH, deamino-NADPH, thio-NADPH, and acetylpyridine-NADPH. Although neither dihydrofolate nor NADP+and its analog form stable complexes withL. caseidihydrofolate reductase, both NADP+and deamino-NADP+interact with enzyme and dihydrofolate to generate stable ternary complexes.