Role of aspartate-37 in determining cofactor specificity and binding in rat liver dihydropteridine reductase.
Role of aspartate-37 in determining cofactor specificity and binding in rat liver dihydropteridine reductase.
复制标题
天冬氨酸 37 在确定大鼠肝脏二氢蝶啶还原酶的辅因子特异性和结合中的作用。
DOI：
10.1016/0003-9861(91)90412-c
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发表时间：
1991
影响因子：
3.9
通讯作者：
Whiteley,JM
中科院分区：
文献类型：
--
作者：
Matthews,DA;Varughese,KI;Skinner,M;Xuong,NH;Hoch,J;Trach,K;Schneider,M;Bray,T;Whiteley,JM
将大鼠二氢蝶啶还原酶（DHPR）全长cDNA与原核表达载体连接，并导入大肠杆菌中。转化的细菌表达二氢蝶啶还原酶免疫反应性蛋白质，并证明quinidazole二氢蝶啶转化为它们的四氢形式。几种重组酶已被纯化至均一，并进行了生化研究，将其特性与大鼠肝酶所表现出的特性进行比较。重组酶和天然酶的最佳反应条件、动力学常数和稳定性相似。结果表明，非突变的重组大鼠DHPR是一个真实的复制品的天然蛋白质和DHPR活性的特点是由一个单一的基因产物，并不需要通过真核细胞的特定修饰。除野生型外，还形成了还原酶的三种特异性诱变形式A-6-V、W-104-F和D-37-I，以及另外的缩写结构。每种产物都表现出还原酶活性，尽管它们对它们的辅因子NADH表现出不同的亲和力，并且对色谱、透析和浓缩的稳定性低于野生型酶。N-末端序列在氨基酸9和36之间含有经典的NADH结合区，Asp 37是结合辅因子所必需的，如D-37-I突变体的解离常数增加约20倍和kcat减少（约43 s− 1，而野生型酶为156 s− 1）所示。结果表明，DHPR辅因子结合位点类似于典型的二核苷酸需要的脱氢酶，如乳酸和肝醇脱氢酶。
Full-length rat dihydropteridine reductase (DHPR) cDNAs have been combined with a prokaryotic expression vector and introduced intoEscherichia coli. Transformed bacteria express dihydropteridine reductase immunoreactive proteins and demonstrate conversion of quinonoid dihydropteridines to their tetrahydro forms. Several recombinant enzymes have been purified to homogeneity and biochemical studies have been carried out comparing their properties with those exhibited by the rat liver enzyme. The optimal reaction conditions, kinetic constants, and stability are similar for the recombinant and naturally occurring enzyme. The results indicate that the nonmutant recombinant rat DHPR is an authentic replica of the natural protein and that the characteristics of DHPR activity are determined by a single gene product and do not require specific modification via the eukaryotic cell. In addition to the wild type, three specific mutagenic forms of the reductase, A-6-V, W-104-F, and D-37-I, and an additional abbreviated structure have also been formed. Each of the products exhibits reductase activity, although they show varied affinities for their cofactor, NADH, and less stability to chromatography, dialysis, and concentration than the wild-type enzyme. The N-terminal sequence contains a classic NADH binding region between amino acids 9 and 36, and Asp 37 is essential for binding the cofactor as is shown by the ~20-fold increase in dissociation constant for the D-37-I mutant and diminishedkcat(~43 s−1compared to 156 s−1for the wild-type enzyme). The results indicate that the DHPR cofactor binding site is similar to typical dinucleotide requiring dehydrogenases such as lactic acid and liver alcohol dehydrogenase.