Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase.
Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase.
复制标题
苯丙氨酸 31 转化为亮氨酸对人二氢叶酸还原酶功能的影响。
DOI：
10.1021/bi00437a020
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Freisheim,JH
中科院分区：
文献类型：
--
作者：
Prendergast,NJ;Appleman,JR;Delcamp,TJ;Blakley,RL;Freisheim,JH
生物化学系，俄亥俄州医学院，CS 10008，Toledo，俄亥俄州43699，生物化学和临床药理学系，St. Jude儿童研究医院，孟菲斯，田纳西州38101，和药理学系，田纳西大学医学院-孟菲斯，孟菲斯，田纳西州38163接收日期：1988年11月29日; 1989年2月23日摘要：寡核苷酸定向定点诱变用于将人重组二氢叶酸还原酶（DHFR）的苯丙氨酸-31转化为亮氨酸。鉴于早期的化学修饰研究，这种取代是令人感兴趣的（Kumar等人，1981）和基于X射线晶体学数据的结构研究（马修斯等人，1985 a，B），该研究表明鸡肝DHFR中的相应残基Tyr-31与二氢叶酸的结合有关。此外，这种特殊的取代允许测试哺乳动物和细菌还原酶之间的蛋白质序列差异的意义在这个位置上的甲氧苄啶的物种选择性。野生型（WT）和突变型（F31 L）酶均通过使用先前描述的异源表达系统表达和纯化（Prendergast等人，1988年）。WT和F31 L对甲氧苄啶的抑制常数（K值）分别为1.00和1.08 µ。因此，在人二氢叶酸还原酶的31位存在苯丙氨酸并不有助于甲氧苄啶的物种选择性。对于突变酶，烟酰胺腺嘌呤二核苷酸磷酸（还原）（NADPH）和二氢叶酸的Km值分别升高了10.8倍和9.4倍，而KmM仅增加了1.8倍。平衡解离常数（KD值），获得了结合的NADPH和二氢叶酸在二元复合物与每种酶。WT和F31 L中NADPH的KO相似，而F31 L中二氢叶酸的KD低43倍。用停流技术测定了二氢叶酸与酶和酶-NADPH复合物的结合速率常数（kon）。单酶法和酶-NADPH法分别为（3.5±0.8）× 108 M ~（-1）s ~（-1）（F31 L）和（2.2±0.3）× 108 M ~（-1）s ~（-1）（WT），酶-NADPH法分别为（0.5±0.1）× 108 M ~（-1）s ~（-1）（F31 L）和（1.5±0.2）× 108 M ~（-1）s ~（-1）（WT）。基于与野生型相比二氢叶酸与F31 L相互作用的这些改变，我们得出结论，Phe-31在平衡条件下直接在底物结合脱辅基酶以及在稳态催化循环期间发生的酶形式中起作用。1.3)是细胞生长和复制所必需的酶。DHFR催化烟酰胺腺嘌呤二核苷酸磷酸（还原型）（NADPH）将二氢叶酸（H2 F）还原为四氢叶酸（H4 F），这是嘌呤、嘧啶和核苷酸生物合成持续进行所必需的过程。这种酶的抑制剂，最值得注意的是抗叶酸化合物甲氨蝶呤（MTX）1和甲氧苄啶（TMP）1，广泛用于治疗肿瘤和感染性疾病的临床环境。甲氧苄啶是一种特别有效的抗菌剂，其能够在比对人（h）1 DHFR产生相同效果所需的浓度低30000-60000倍的浓度下对大肠杆菌DHFR产生50%的抑制（Burchall & Hitchings，1965; Li等人，1982年）。TMP抑制DHFR的物种选择性的分子基础已经被广泛综述（马修斯等人，1985 a，B; Birdsall等人，1983年）。使用改进的X…
Department of Biochemistry, Medical College of Ohio, CS 10008, Toledo, Ohio 43699, Department of Biochemical and Clinical Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38101, and Department of Pharmacology, University of Tennessee College of Medicine—Memphis, Memphis, Tennessee 38163 Received November 29, 1988; Revised Manuscript Received February 23, 1989 abstract: Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a, b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified byusing a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (K¡ values) for trimethoprim were 1.00 and 1.08 µ for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced)(NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the KmM increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KO for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and en-zyme-NADPH complexes were measured by stopped-flow techniques. These values are (3.5±0.8) X 108 M_I s"* 1 (F31L) and (2.2±0.3) X 108 M_1 s_1 (WT) with enzyme alone and (0.5±0.1) X 108 M" 1 s_1 (F31L) and (1.5±0.2) X 108 M_1 s_1 (WT) with enzyme-NADPH. On the basis of these alterations in the interaction of dihydrofolate with F31L when compared to wild type, we conclude that Phe-31 functions directly in substrate binding to apoenzyme under equilibrium conditions as well as to enzyme forms occurring during steady-state catalytic cycling.Dihydrofolate reductase (DHFR) 1 (5, 6, 7, 8-tetrahydro-folate: NADP+ oxidoreductase, EC 1.5. 1.3) is an enzyme es-sential for the growth and replication of cells. DHFR catalyzes the nicotinamide adenine dinucleotide phosphate (reduced)(NADPH)’-dependent reduction of dihydrofolate (H2F)· to tetrahydrofolate (H4F),'a process necessary for the continuation of purine, pyrimidine, and nucleotide biosynthesis. In-hibitors of this enzyme, most notably the anti-folate compounds methotrexate (MTX) 1 and trimethoprim (TMP), 1 are used extensively in clinical settings for the treatment of neoplastic and infectious disorders. Trimethoprim is a particularly potent antibacterial agent capable of producing 50% inhibition of Escherichia coli DHFR at concentrations 30000-60000 times lower than needed to produce the same effect on human (h) 1 DHFR (Burchall & Hitchings, 1965; Li et al., 1982). The molecular basis for the species selectivity of DHFR inhibition by TMP has been extensively reviewed (Matthews et al., 1985a, b; Birdsall et al., 1983). Using refined X …