Mapping the conformational itinerary of β-glycosidases by X-ray crystallography

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–O-5 bond. This demands planarity of C-5, O-5, C-1 and C-2 at or near the transition state. It is widely, but incorrectly, assumed that the transition state must be ⁴H₃ (half-chair). The transition-state geometry is equally well supported, for pyranosides, by both the ⁴H₃ and ³H₄ half-chair and ^2,5B and B_2,5 boat conformations. A number of retaining β-glycosidases acting on gluco-configured substrates have been trapped in Michaelis and covalent intermediate complexes in ¹S₃ (skew-boat) and ⁴C₁ (chair) conformations, respectively, pointing to a ⁴H₃-conformed transition state. Such a ⁴H₃ conformation is consistent with the tight binding of ⁴E- (envelope) and ⁴H₃-conformed transition-state mimics to these enzymes and with the solution structures of compounds bearing an sp² hybridized anomeric centre. Recent work reveals a ¹S₅ Michaelis complex for β-mannanases which, together with the ⁰S₂ covalent intermediate, strongly implicates a B_2,5 transition state for β-mannanases, again consistent with the solution structures of manno-configured compounds bearing an sp² anomeric centre. Other enzymes may use different strategies. Xylanases in family GH-11 reveal a covalent intermediate structure in a ^2,5B conformation which would also suggest a similarly shaped transition state, while ²S₀-conformed substrate mimics spanning the active centre of inverting cellulases from family GH-6 may also be indicative of a ^2,5B transition-state conformation. Work in other laboratories on both retaining and inverting α-mannosidases also suggests non-⁴H₃ 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.