Inversion of the roles of the nucleophile and acid/base catalysts in the covalent binding of epoxyalkyl xyloside inhibitor to the catalytic glutamates of endo-1,4-beta-xylanase (XYNII): a molecular dynamics study

X-Ray crystal structures have revealed that 2,3-epoxypropyl-beta-D-xyloside reacts with endo-1,4-beta-xylanase (XYNII) by forming a covalent bond with Glu86. In contrast, 3,4-epoxybutyl-beta-D-xyloside forms a covalent bond w ith Glu177. In the normal enzyme reaction Glu86 acts as the catalytic nucle ophile and Glu177 as the acid/base catalyst. To rationalize the observed re activity of the two mechanism-based inhibitors, we carried out eight 300 ps molecular dynamics simulations for different enzyme-inhibitor complexes. S imulations were done for both stereo isomers (R and S) of the inhibitors an d for enzyme in which the protonation state of the nucleophile and acid/bas e catalyst was normal (Glu86 charged, Glu177 neutral) and in which the role s of the catalytic residues were reversed (Glu86 neutral, Glu177 charged), The number of reactive conformations found in each simulation was used to p redict the reactivity of epoxy inhibitors. The conformation was considered to be a reactive one when at the same time (i) the proton of the catalytic acid was close (<2.9/3.4/3.9 Angstrom) to the oxirane oxygen of the inhibit or, (ii) the nucleophile was close to the terminal carbon of the oxirane gr oup (<3.4/3.9/4.4 Angstrom) and (iii) the nucleophile approached the termin al carbon from a reactive angle (<30/45/60 degrees from an ideal attack ang le). On the basis of the number of reactive conformations, 2,3-epoxypropyl- beta-D-xyloside was predicted to form a covalent bond with Glu86 and 3,4-ep oxybutyl-beta-D-xyloside with Glu177, both in agreement with the experiment . Thus, the MD simulations and the X-ray structures indicate that in the co valent binding of 3,4-epoxybutyl-beta-D-xyloside the roles of the catalytic glutamates of XYNII are reversed from that of the normal enzyme reaction.