Role of Lys-32 residues in R67 dihydrofolate reductase probed by asymmetric mutations
Role of Lys-32 residues in R67 dihydrofolate reductase probed by asymmetric mutations
复制标题
DOI：
10.1074/jbc.m404484200
复制
发表时间：
2004-11-05
影响因子：
4.8
通讯作者：
Howell, EE
中科院分区：
文献类型：
--
作者：
Hicks, SN;Smiley, RD;Howell, EE
R67二氢叶酸还原酶（R67 DHFR）是一种由r质粒编码的新型蛋白，可对抗生素甲氧苄啶产生耐药性。这种同四聚体酶具有222个对称性，这对单个活性位点孔施加了许多限制，包括结合其配体二氢叶酸（DHF）和NADPH的“一个位点适合两个位点”策略。先前的研究揭示了盐对结合和催化的影响（Hicks, s.n ., Smiley， R. D., Hamilton, j.b ., and Howell, e.e. (2003) Biochemistry 42,10569 -10578），但是参与DHF负电荷尾巴离子接触的一个或多个残基以及NADPH中的磷酸基团尚未确定。一些研究预测Lys-32残基参与其中，然而该残基的突变破坏了R67 DHFR同型四聚体的稳定性。为了研究Lys-32在结合和催化中的作用，我们利用了不对称的K32M突变。为了产生不对称，个体突变被添加到四个框内基因拷贝的串联阵列中。这些研究表明，一个K32M突变的耐受性相当好，而两个突变的增加则有不同的影响。两个双突变体，K32M: 1 + 2和K32M: 1 + 4，将突变放置在孔的两侧，减少k（cat）。然而，第三个双突变体K32M: 1 + 3，在相同的半孔上放置了两个突变，与母体酶相比，k（cat）增强了4- 5倍，尽管是以较弱的配体结合为代价。由于该双突变序列的k(cat)/ k -m值相似，这些突变似乎揭示了某种程度的非生产性结合。这种非生产性结合模式可能源于离子相互作用的形成，必须打破离子相互作用才能进入过渡态。K32M:1 + 3突变体数据表明，这种相互作用是Lys-32与二氢叶酸的带电尾部之间的离子相互作用。这种不寻常的催化情景是由施加在单个活性位点孔上的222对称性引起的。
R67 dihydrofolate reductase ( R67 DHFR) is a novel protein encoded by an R-plasmid that confers resistance to the antibiotic, trimethoprim. This homotetrameric enzyme possesses 222 symmetry, which imposes numerous constraints on the single active site pore, including a "one-site-fits-both" strategy for binding its ligands, dihydrofolate (DHF) and NADPH. Previous studies uncovered salt effects on binding and catalysis ( Hicks, S. N., Smiley, R. D., Hamilton, J. B., and Howell, E. E. ( 2003) Biochemistry 42, 10569-10578), however the one or more residues that participate in ionic contacts with the negatively charged tail of DHF as well as the phosphate groups in NADPH were not identified. Several studies predict that Lys-32 residues were involved, however mutations at this residue destabilize the R67 DHFR homotetramer. To study the role of Lys-32 in binding and catalysis, asymmetric K32M mutations have been utilized. To create asymmetry, individual mutations were added to a tandem array of four in-frame gene copies. These studies show one K32M mutation is tolerated quite well, whereas addition of two mutations has variable effects. Two double mutants, K32M: 1 + 2 and K32M: 1 + 4, which place the mutations on opposite sides of the pore, reduce k(cat). However a third double mutant, K32M: 1 + 3, that places two mutations on the same half pore, enhances k(cat) 4- to 5-fold compared with the parent enzyme, albeit at the expense of weaker binding of ligands. Because the k(cat)/K-m values for this double mutant series are similar, these mutations appear to have uncovered some degree of non-productive binding. This non-productive binding mode likely arises from formation of an ionic interaction that must be broken to allow access to the transition state. The K32M:1 + 3 mutant data suggest this interaction is an ionic interaction between Lys-32 and the charged tail of dihydrofolate. This unusual catalytic scenario arises from the 222 symmetry imposed on the single active site pore.