Vaaje-Kolstad Gustav, Houston Douglas R, Rao Francesco V, Peter Martin G, Synstad Bjørnar, van Aalten Daan M F, Eijsink Vincent G H
Department of Chemistry and Biotechnology, Agricultural University of Norway, PO Box 5040, N-1432 Aas, Norway.
Biochim Biophys Acta. 2004 Jan 14;1696(1):103-11. doi: 10.1016/j.bbapap.2003.09.014.
Catalysis by ChiB, a family 18 chitinase from Serratia marcescens, involves a conformational change of Asp142 which is part of a characteristic D(140)XD(142)XE(144) sequence motif. In the free enzyme Asp142 points towards Asp140, whereas it rotates towards the catalytic acid, Glu144, upon ligand binding. Mutation of Asp142 to Asn reduced k(cat) and affinity for allosamidin, a competitive inhibitor. The X-ray structure of the D142N mutant showed that Asn142 points towards Glu144 in the absence of a ligand. The active site also showed other structural adjustments (Tyr10, Ser93) that had previously been observed in the wild-type enzyme upon substrate binding. The X-ray structure of a complex of D142N with allosamidin, a pseudotrisaccharide competitive inhibitor, was essentially identical to that of the wild-type enzyme in complex with the same compound. Thus, the reduced allosamidin affinity in the mutant is not caused by structural changes but solely by the loss of electrostatic interactions with Asp142. The importance of electrostatics was further confirmed by the pH dependence of catalysis and allosamidin inhibition. The pH-dependent apparent affinities for allosamidin were not correlated with k(cat), indicating that it is probably better to view the inhibitor as a mimic of the oxazolinium ion reaction intermediate than as a transition state analogue.
来自粘质沙雷氏菌的18家族几丁质酶ChiB的催化作用涉及Asp142的构象变化，Asp142是特征性D(140)XD(142)XE(144)序列基序的一部分。在游离酶中，Asp142指向Asp140，而在配体结合后，它会转向催化酸Glu144。将Asp142突变为Asn会降低k(cat)以及对竞争性抑制剂别洛沙米定的亲和力。D142N突变体的X射线结构表明，在没有配体的情况下，Asn142指向Glu144。活性位点还显示出其他结构调整（Tyr10、Ser93），这些调整在野生型酶与底物结合时也曾被观察到。D142N与假三糖竞争性抑制剂别洛沙米定的复合物的X射线结构与野生型酶与同一化合物的复合物的结构基本相同。因此，突变体中别洛沙米定亲和力的降低不是由结构变化引起的，而仅仅是由于与Asp142的静电相互作用丧失。催化作用和别洛沙米定抑制作用的pH依赖性进一步证实了静电作用的重要性。别洛沙米定的pH依赖性表观亲和力与k(cat)不相关，这表明将该抑制剂视为恶唑啉离子反应中间体的模拟物可能比视为过渡态类似物更好。
