Mapping the conformational itinerary of β-glycosidases by X-ray crystallography
Mapping the conformational itinerary of β-glycosidases by X-ray crystallography
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DOI：
10.1042/bst0310523
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发表时间：
2003-06-01
影响因子：
3.9
通讯作者：
Zechel, DL
中科院分区：
文献类型：
--
作者：
Davies, GJ;Ducros, VMA;Zechel, DL
利用不同的糖苷酶沿着反应途径的构象议程已被映射的X射线晶体学。在糖苷的酶促水解过程中形成的过渡态的特征在于涉及跨C-1-0-5键的离域的强氧碳离子样特征。这要求C-5、0-5、C-1和C-2在过渡态或其附近具有平面性。人们普遍认为过渡态必须是H-4（3）（半椅），但这是错误的。对于吡喃糖苷，H-4（3）和H-3（4）半椅型以及B-2，B-5和B-2，B-5船型构象同样支持过渡态几何结构。许多作用于葡萄糖构型底物的保留β-糖苷酶已分别以S-1（3）（斜船）和C-4（1）（椅）构象被捕获在米氏和共价中间体复合物中，指向H-4（3）-顺应的过渡态。这种H-4（3）构象与E-4-（包膜）和H-4（3）-顺应的过渡态模拟物与这些酶的紧密结合以及带有sp（2）杂化异头中心的化合物的溶液结构一致。最近的工作揭示了β-甘露聚糖酶的S-1（5）米氏络合物，其与S-0（2）共价中间体一起强烈暗示β-甘露聚糖酶的B2，s过渡态，再次与带有sp（2）异头中心的甘露糖构型化合物的溶液结构一致。其他酶可以使用不同的策略。GH-11家族中的木聚糖酶显示了B-2，B-5构象的共价中间结构，这也表明了类似形状的过渡态，而跨越来自GH-6家族的转化纤维素酶的活性中心的S-2（0）-顺应的底物模拟物也可以指示B-2，B-5过渡态构象。其他实验室对保留和转化α-甘露糖苷酶的研究也表明这些医学上重要的酶具有非H-4（3）过渡态。酶复合物的三维结构现在应该能够驱动特定酶的过渡态模拟物的设计，而不是通用的或仅仅是偶然的。
The conformational agenda harnessed by different glycosidases along the reaction pathway has been mapped by X-ray crystallography. The transition state(s) formed during the enzymic hydrolysis of glycosides features strong oxocarbenium-ion-like character involving delocalization across the C-1-0-5 bond. This demands planarity of C-5, 0-5, C-1 and C-2 at or near the transition state. It is widely, but incorrectly, assumed that the transition state must be H-4(3) (half-chair). The transition-state geometry is equally well supported, for pyranosides, by both the H-4(3) and H-3(4) half-chair and B-2,B-5 and B-2,B-5 boat conformations. A number of retaining,beta-glycosidases acting on gluco-configured substrates have been trapped in Michaelis and covalent intermediate complexes in S-1(3) (skew-boat) and C-4(1) (chair) conformations, respectively, pointing to a H-4(3)-conformed transition state. Such a H-4(3) conformation is consistent with the tight binding of E-4-(envelope) and H-4(3)-conformed transition-state mimics to these enzymes and with the solution structures of compounds bearing an sp(2) hybridized anomeric centre. Recent work reveals a S-1(5) Michaelis complex for beta-mannanases which, together with the S-0(2) covalent intermediate, strongly implicates a B2,s transition state for beta-mannanases, again consistent with the solution structures of manno-configured compounds bearing an sp(2) anomeric centre. Other enzymes may use different strategies. Xylanases in family GH-11 reveal a covalent intermediate structure in a B-2,B-5 conformation which would also suggest a similarly shaped transition state, while S-2(0)-conformed substrate mimics spanning the active centre of inverting cellulases from family GH-6 may also be indicative of a B-2,B-5 transition-state conformation. Work in other laboratories on both retaining and inverting alpha-mannosidases also suggests non-H-4(3) transition states for these medically important enzymes. Three-dimensional structures of enzyme complexes should now be able to drive the design of transition-state mimics that are specific for given enzymes, as opposed to being generic or merely fortuitous.