Initial catalytic events in class 3 aldehyde dehydrogenase: MM and QM/MM simulations.

A novel enzyme mechanism has been predicted by computer simulations for formation of the thiohemiacetal intermediate in the rat ALDH3A1 enzyme. We used molecular mechanics simulations to study the atomic details of substrate binding and quantum mechanical/molecular mechanical methods to study the Cys-243 thiolate attack on benzaldehyde (BA) substrate. BA was found to produce more reactive conformers when aligned for formation of the tetrahedral thiohemiacetal in the R-configuration. In addition, the sulfhydryl proton was seen to be important for initial binding of the substrate. Finally, the free energy differences between forming a thiohemiacetal oxyanion intermediate versus forming a neutral thiohemiacetal intermediate where a proton is donated to the intermediate from the surroundings strongly favor the latter. Our results suggest that the proton donor is the amide proton from the Cys-243 backbone supported by interactions with Lys-235.