Smith S L, Burchall J J
Proc Natl Acad Sci U S A. 1983 Aug;80(15):4619-23. doi: 10.1073/pnas.80.15.4619.
The alpha epimers of pyridine nucleotides are almost totally inactive as reductants in dehydrogenase reactions. In contrast, the R plasmid R67-specified dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) isolated from trimethoprim-resistant Escherichia coli utilized alpha-NADPH and alpha-NADH in addition to the "normal" beta-epimers. The enzymes from bacterial and mammalian sources used only beta-NADPH and beta-NADH. THe Km value for alpha-NADPH (16 microM) was 4-fold greater than that for beta-NADPH (4 microM), while the maximal velocity of the alpha-NADPH-catalyzed reaction was 70% of that seen with the beta-NADPH. beta-NADP+ and alpha-NADP+ were competitive inhibitors of the R67 enzyme. Pyridine nucleotide analogues such as deamino- and acetyl-NADPH were used readily by bacterial, plasmid, and mammalian enzymes, whereas thio-NADPH was used only by the plasmid enzyme. These data suggest that the enzyme from R plasmid R67 possesses a pyridine nucleotide binding site different from that of other dihydrofolate reductases and dehydrogenases.
吡啶核苷酸的α差向异构体在脱氢酶反应中作为还原剂几乎完全没有活性。相比之下，从耐甲氧苄啶的大肠杆菌中分离出的R质粒R67指定的二氢叶酸还原酶（5,6,7,8-四氢叶酸：NADP +氧化还原酶，EC 1.5.1.3）除了利用“正常”的β差向异构体之外，还利用α-NADPH和α-NADH。来自细菌和哺乳动物来源的酶仅使用β-NADPH和β-NADH。α-NADPH的Km值（16μM）比β-NADPH的Km值（4μM）大4倍，而α-NADPH催化反应的最大速度是β-NADPH催化反应最大速度的70％。β-NADP +和α-NADP +是R67酶的竞争性抑制剂。细菌、质粒和哺乳动物的酶很容易利用吡啶核苷酸类似物，如脱氨基和乙酰基-NADPH，而硫代-NADPH仅被质粒酶利用。这些数据表明，R质粒R67的酶具有与其他二氢叶酸还原酶和脱氢酶不同的吡啶核苷酸结合位点。
