Guanidine derivatives rescue the Arg418Ala mutation of Tritrichomonas foetus IMP dehydrogenase
Guanidine derivatives rescue the Arg418Ala mutation of Tritrichomonas foetus IMP dehydrogenase
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DOI：
10.1021/bi051603w
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发表时间：
2005-12-20
期刊：
影响因子：
2.9
通讯作者：
Hedstrom, L
中科院分区：
文献类型：
--
作者：
Schlippe, YVG;Hedstrom, L
IMP脱氢酶（IMPDH）催化肌苷5'-单磷酸（IMP）氧化为黄苷5'-单磷酸（XMP）和NAD+的还原。该反应涉及E-XMP * 共价中间体的形成; E-XMP * 中间体的水解是限速的，并且需要酶采取闭合构象。Arg418似乎充当活化水用于水解反应的碱[Guillen-Schlippe，Y.五、和Hedstrom，L.（2005）Biochemistry 44，11700 - 11707]。Arg418的去质子化也稳定了闭合构象。在这里，我们表明，胍衍生物救援的Arg418Ala变体的活性。胺和咪唑不能拯救。救援反应似乎是饱和的，K-R的值范围从40到400 mM。最好的救援剂的k值（救援）接近野生型酶反应的k值（cat）。胍衍生物还可以拯救Arg 418 Ala/Tyr 419 Phe变体的活性。多重抑制剂实验表明，胍衍生物不恢复开放和封闭构象之间的平衡。因此，救援剂必须加速E-XMP * 中间体的水解。救援反应的速率随着pH的增加而增加，这与反应涉及中性胍的假设一致。在低浓度的救援剂下观察到溶剂D2O同位素效应，这与质子从水中的限速转移一致。kcat（救援）/K-R（碱）的值与胍衍生物的pK（α）相关（布朗斯台德系数β类似于1）。这些结果表明，质子从水转移到胍是几乎完全的过渡态。
IMP dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) and the reduction of NAD+. The reaction involves formation of an E-XMP* covalent intermediate; hydrolysis of the E-XMP* intermediate is rate-limiting and requires the enzyme to adopt a closed conformation. Arg418 appears to act as the base that activates water for the hydrolysis reaction [Guillen-Schlippe, Y. V., and Hedstrom, L. (2005) Biochemistry 44, 11700-11707]. Deprotonation of Arg418 also stabilizes the closed conformation. Here we show that guanidine derivatives rescue the activity of the Arg418Ala variant. Amines and imidazole do not rescue. The rescue reaction appears to be saturable, with the values of K-R ranging from 40 to 400 mM. The value of k(rescue) for the best rescue agents approaches the value of k(cat) for the reaction of the wild-type enzyme. Guanidine derivatives also rescue the activity of the Arg418Ala/Tyr419Phe variant. Multiple-inhibitor experiments suggest that the guanidine derivatives do not restore the equilibrium between open and closed conformations. Therefore, rescue agents must accelerate the hydrolysis of the E-XMP* intermediate. The rate of the rescue reaction increases with an increase in pH, consistent with the hypothesis that the reaction involves neutral guanidine. A solvent D2O isotope effect is observed at low concentrations of the rescue agent, consistent with rate-limiting transfer of a proton from water. The value of kcat (rescue)/K-R(base) correlates with the pK(a) of the guanidine derivative (Bronsted coefficient beta similar to 1). These results suggest that proton transfer from water to guanidine is almost complete in the transition state.