Park H, Zhuang P, Nichols R, Howell E E
Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996-0840, USA.
J Biol Chem. 1997 Jan 24;272(4):2252-8. doi: 10.1074/jbc.272.4.2252.
R67 dihydrofolate reductase (DHFR) is encoded by an R-plasmid, and expression of this enzyme in bacteria confers resistance to the antibacterial drug, trimethoprim. This DHFR variant is not homologous in either sequence or structure with chromosomal DHFRs. The crystal structure of tetrameric R67 DHFR indicates a single active site pore that traverses the length of the molecule (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Nat. Struct. Biol. 2, 1018-1025). A pH profile of enzyme activity in R67 DHFR displays an acidic pKa that is protein concentration-dependent. This pKa describes dissociation of active tetramer into two relatively inactive dimers upon protonation of His-62 and the symmetry-related His-162, His-262, and His-362 residues at the dimer-dimer interfaces. Construction of an H62C mutation results in stabilization of the active tetramer via disulfide bond formation at the dimer-dimer interfaces. The oxidized, tetrameric form of H62C R67 DHFR is quite active at pH 7, and a pH profile displays increasing activity at low pH. These results indicate protonated dihydrofolate (pKa = 2.59) is the productive substrate and that R67 DHFR does not possess a proton donor.
R67二氢叶酸还原酶（DHFR）由一个R质粒编码，该酶在细菌中的表达赋予了对抗菌药物甲氧苄啶的抗性。这种DHFR变体在序列或结构上与染色体DHFR均无同源性。四聚体R67 DHFR的晶体结构显示有一个贯穿分子全长的单一活性位点孔道（纳拉亚纳，N.，马修斯，D. A.，豪厄尔，E. E.，和阮，N.-H.（1995年）《自然结构生物学》2，1018 - 1025）。R67 DHFR酶活性的pH曲线显示出一个酸性pKa，它依赖于蛋白质浓度。这个pKa描述了在二聚体 - 二聚体界面处，当His - 62以及对称相关的His - 162、His - 262和His - 362残基质子化时，活性四聚体解离为两个相对无活性的二聚体。构建H62C突变体可通过在二聚体 - 二聚体界面处形成二硫键来稳定活性四聚体。氧化态的H62C R67 DHFR四聚体在pH 7时活性相当高，其pH曲线显示在低pH下活性增加。这些结果表明质子化二氢叶酸（pKa = 2.59）是有效的底物，并且R67 DHFR不具有质子供体。
