Viladot J L, de Ramon E, Durany O, Planas A
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona, Spain.
Biochemistry. 1998 Aug 11;37(32):11332-42. doi: 10.1021/bi980586q.
The role of the key catalytic residues Glu134 and Glu138 in the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis is probed by a chemical rescue methodology based on enzyme activation of inactive mutants by the action of added nucleophiles. While Glu134 was proposed as the catalytic nucleophile on the basis of affinity labeling experiments, no functional proof supported the assignment of Glu138 as the general acid-base catalyst. Alanine replacements are prepared by site-directed mutagenesis to produce the inactive E138A and E134A mutants. Addition of azide reactivates the mutants in a concentration-dependent manner using an activated 2, 4-dinitrophenyl glycoside substrate. The chemical rescue operates by a different mechanism depending on the mutant as deduced from 1H NMR monitoring and kinetic analysis of enzyme reactivation. E138A yields the beta-glycosyl azide product arising from nucleophilic attack of azide on the glycosyl-enzyme intermediate, thus proving that Glu138 is the general acid-base residue. Azide activates the deglycosylation step (increasing kcat), but it also has a large effect on a previous step (as seen by the large decrease in KM, the increase in kcat/KM, and the pH dependence of activation), probably increasing the rate of glycosylation through Bronsted acid catalysis by enzyme-bound HN3. By contrast, azide reactivates the E134A mutant through a single inverting displacement to give the alpha-glycosyl azide product, consistent with Glu134 being the catalytic nucleophile. Formate as an exogenous nucleophile has no effect on the E138A mutant, whereas it is a better activator of E134A than azide. Although the reaction yields the normal hydrolysis product, a transient compound was detected by 1H NMR, tentatively assigned to the alpha-glycosyl formate adduct. This is the first case where a nonmodified sugar gives a long-lived covalent intermediate that mimics the proposed glycosyl-enzyme intermediate of retaining glycosidases.
通过一种化学拯救方法，基于添加亲核试剂对无活性突变体的酶激活作用，探究了地衣芽孢杆菌保留型1,3 - 1,4-β-葡聚糖酶中关键催化残基Glu134和Glu138的作用。虽然基于亲和标记实验提出Glu134为催化亲核试剂，但没有功能证据支持将Glu138指定为广义酸碱催化剂。通过定点诱变制备丙氨酸替代物，以产生无活性的E138A和E134A突变体。使用活化的2,4 - 二硝基苯基糖苷底物，叠氮化物的添加以浓度依赖的方式使突变体重激活。根据1H NMR监测和酶再激活的动力学分析推断，化学拯救根据突变体的不同通过不同机制起作用。E138A产生叠氮化物对糖基酶中间体进行亲核攻击产生的β-糖基叠氮产物，从而证明Glu138是广义酸碱残基。叠氮化物激活去糖基化步骤（增加kcat），但它也对前一步骤有很大影响（如KM大幅降低、kcat/KM增加以及激活的pH依赖性所示），可能是通过酶结合的HN3的布朗斯特酸催化增加糖基化速率。相比之下，叠氮化物通过单一的构型翻转取代使E134A突变体重激活，产生α-糖基叠氮产物，这与Glu134是催化亲核试剂一致。甲酸盐作为外源性亲核试剂对E138A突变体没有影响，而它对E134A的激活作用比叠氮化物更好。虽然反应产生正常的水解产物，但通过1H NMR检测到一种瞬态化合物，初步确定为α-糖基甲酸酯加合物。这是首次发现未修饰的糖产生一种长寿命的共价中间体，该中间体模拟了保留型糖苷酶提出的糖基酶中间体。
