Structural and biochemical evidence for a boat-like transition state in β-mannosidases

Enzyme inhibition through mimicry of the transition state is a major area for the design of new therapeutic agents. Emerging evidence suggests that many retaining glycosidases that are active on alpha- or beta-mannosides harness unusual B-2,B-5 (boat) transition states. Here we present the analysis of 25 putative beta-mannosidase inhibitors, whose K-i values range from nanomolar to millimolar, on the Bacteroides thetaiotaomicron b-mannosidase BtMan2A. B-2,B-5 or closely related conformations were observed for all tightly binding compounds. Subsequent linear free energy relationships that correlate log K-i with log K-m/K-cat for a series of active center variants highlight aryl-substituted mannoimidazoles as powerful transition state mimics in which the binding energy of the aryl group enhances both binding and the degree of transition state mimicry. Support for a B-2,B-5 transition state during enzymatic beta-mannosidase hydrolysis should also facilitate the design and exploitation of transition state mimics for the inhibition of retaining alpha-mannosidases-an area that is emerging for anticancer therapeutics.