Changing Enzymic Reaction Mechanisms by Mutagenesis: Conversion of a Retaining Glucosidase to an Inverting Enzyme
Changing Enzymic Reaction Mechanisms by Mutagenesis: Conversion of a Retaining Glucosidase to an Inverting Enzyme
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DOI：
10.1021/ja00104a060
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发表时间：
1994-12
影响因子：
15
通讯作者：
Qingping Wang;R. Graham;D. Trimbur;R. Warren;S. Withers
中科院分区：
文献类型：
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作者：
Qingping Wang;R. Graham;D. Trimbur;R. Warren;S. Withers
糖苷酶采用两种独立且不同的机制。1-4在一组酶中，直接置换导致异头构型的反转（方案1A）。在另一组中，通过涉及糖基-酶中间体的双置换机制保留异头构型（方案1B）。虽然这两种机制是非常不同的，但有显着的相似之处：都涉及oxocarbenium离子样过渡态，* 12345，都涉及一对羧酸，在这两种情况下具有不同的作用。在“反相器”中，一个充当酸催化剂，另一个充当碱催化剂，而在“保留器”中，一个充当酸/碱催化剂，另一个充当亲核试剂/离去基团。此外，这两个残基在反转中比在保留糖苷酶中相距更远，以允许水分子的干预。保留的α-和β-糖苷酶的平均分离分别为4.8±0.5和5.3±0.2 μ m，而转化的α-和β-糖苷酶的平均分离分别为9.0±1.0和9.5 μ m。[6]类似的过渡态提高了通过突变改变分离而将酶从一种机制转换为另一种机制的可能性。本文描述了这样的转换。
Glycosidases employ two separate and distinct mechanisms. 1-4 In one set of enzymes, direct displacement leads to netinversion of anomeric configuration (Scheme 1A). In the other set, anomeric configuration is retained via a double displacement mechanism involving a glycosyl—enzyme intermediate (Scheme IB). While the two mechanisms are quite distinct, there are significant similarities: both involve oxocarbenium ion-like transition states,* 12345 and both involve a pair of carboxylic acids, which have different roles in thetwo cases. In “inverters” one functions as an acid catalyst and the other as a base catalyst, whereas in “retainers” one functions as an acid/base catalyst and the other as a nucleophile/leaving group. Furthermore, the two residues are farther apart in the inverting than in the retaining glycosidases to allow the intervention of a water molecule. The average separation in retaining a-and/S-gly-cosidases is 4.8±0.5 and 5.3±0.2 Á, respectively, but in the inverting a-and/5-glycosidases it is 9.0±1.0 and 9.5 Á, respectively. 6 The similar transition states raises the possibility of converting an enzyme from one mechanism to the other by changing the separation by mutation. This paper describes such a conversion.