The comparative interaction of quinonoid (6R)-dihydrobiopterin and an alternative dihydropterin substrate with wild-type and mutant rat dihydropteridine reductases.
The comparative interaction of quinonoid (6R)-dihydrobiopterin and an alternative dihydropterin substrate with wild-type and mutant rat dihydropteridine reductases.
复制标题
醌类 (6R)-二氢生物蝶呤和替代二氢蝶呤底物与野生型和突变型大鼠二氢蝶啶还原酶的比较相互作用。
DOI：
10.1021/bi970585i
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Whiteley,JM
中科院分区：
文献类型：
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作者：
Kiefer,PM;Grimshaw,CE;Whiteley,JM
利用天然底物醌类(6R)-l-红双氢生物蝶呤（qBH2）和替代底物醌类6,7-二甲基双氢蝶啶（q-6,7- dimepth2），测定了野生型二氢蝶啶还原酶（EC 1.6.99.7）和Ala133Ser、Lys150Gln、Tyr146His、Tyr146Phe单突变型、Tyr146Phe/Ala133Ser和Tyr146Phe/Lys150Gln双突变型酶的动力学参数和一次氘同位素效应。Tyr146或Lys150的突变导致两种蝶呤底物的动力学参数和同位素效应发生显著变化，证实了这些残基在酶介导的氢化物转移中起着关键作用。相比之下，Ala133Ser突变体与野生型酶几乎没有区别。然而，观察到的变化在两种蝶呤基质中是完全不同的。因此，在一系列突变体中，q-6,7- dimepth2的kcat值从野生型酶的150 s-1降低到Tyr146Phe/Lys150Gln双突变体的活性基本为零。相反，在相同的突变体中，qbh2的kcat值比野生型的23 s-1增加了3 - 11倍。对于这两种pterin底物，在Tyr146或Lys150突变后，km （KPt）增加了几个数量级，使用qBH2时，km （KPt）的相对增加幅度更大。在Tyr146和Lys150突变体中观察到的kcat（Dkcat）和kcat/KPt（D(kcat/KPt)）的显著初生氘同位素效应根据所使用的蝶呤底物而变化，最大值可达5.5−6。对于qBH2，其edkcat <Dkcat/ kpt一致被观察到，速率决定步骤归因于四氢蝶呤产物的释放。对于q-6,7- dimepth2，在所有情况下，Dkcat=Dkcat/KPt，催化可能受到在氢化物转移之前发生的异构化步骤的限制。将qbh2与二元E:NADH复合物对接的建模研究为观察到的两种蝶呤底物之间的差异提供了结构上的基本原理。天然底物qBH2对酶活性位点表现出更高的亲和力，可能是由于底物的二羟丙基侧链与含有Asn186、Ser189和Met190残基的极性环相互作用所致。该环在三维结构中的位置与短链脱氢酶/还原酶（SDR）家族的其他成员（包括二氢蝶啶还原酶）推定的底物结合环一致。
Kinetic parameters and primary deuterium isotope effects have been determined for wild-type dihydropteridine reductase (EC 1.6.99.7) and the Ala133Ser, Lys150Gln, Tyr146His, Tyr146Phe single, and Tyr146Phe/Ala133Ser and Tyr146Phe/Lys150Gln double mutant enzyme forms using the natural substrate, quinonoid (6R)-l-erythro-dihydrobiopterin (qBH2) and an alternate substrate, quinonoid 6,7-dimethyldihydropteridine (q-6,7-diMePtH2). Mutation at either Tyr146 or Lys150 resulted in pronounced changes in kinetic parameters and isotope effects for both pterin substrates, confirming a critical role for these residues in enzyme-mediated hydride transfer. By contrast, the Ala133Ser mutant was practically indistinguishable from wild-type enzyme. The changes observed, however, were quite different for the two pterin substrates. Thus,kcatfor q-6,7-diMePtH2decreased across the series of mutants from a value of 150 s-1for wild-type enzyme to essentially zero activity for the Tyr146Phe/Lys150Gln double mutant. Conversely,kcatfor qBH2increased 3−11-fold across the same series of mutants from the wild-type value of 23 s-1. For both pterin substrates, theKm(KPt) increased several orders of magnitude upon mutation of Tyr146 or Lys150, with the greater relative increase using qBH2. Significant primary deuterium isotope effects onkcat(Dkcat) andkcat/KPt(D(kcat/KPt)) observed for the Tyr146 and Lys150 mutants varied depending on the pterin substrate used and ranged up to a maximum value of 5.5−6. For qBH2, whereDkcat<Dkcat/KPtwas consistently observed, the rate determining step is ascribed to release of the tetrahydropterin product. For q-6,7-diMePtH2, where in all casesDkcat=Dkcat/KPt, catalysis is probably limited by an isomerization step occurring prior to hydride transfer. Modeling studies in which qBH2was docked into the binary E:NADH complex provide a structural rationale for the observed differences between the two pterin substrates. The natural substrate, qBH2, displays a higher affinity for the enzyme active site, presumably due to interaction of the dihydroxypropyl side chain of the substrate with a polar loop of residues containing Asn186, Ser189, and Met190. The location of this loop within the three-dimensional structure is consistent with putative substrate binding loops for other members of the short chain dehydrogenase/reductase (SDR) family, which includes dihydropteridine reductase.