Role of catalytic residues in enzymatic mechanisms of homologous ketosteroid isomerases

Ketosteroid isomerase (KSI) is one of the most proficient enzymes catalyzin g an allylic isomerization reaction at a diffusion-controlled rate. In this study of KSI, we have detailed the structures of its active site, the role of various catalytic residues, and have explained the origin of the its fa st reactivity by carrying out a detailed investigation of the enzymatic rea ction mechanism. This investigation included the X-ray determination of 15 crystal structures of two homologous enzymes in free and complexed states ( with inhibitors) and extensive ab initio calculations of the interactions b etween the active sites and the reaction intermediates. The catalytic resid ues, through short strong hydrogen bonds, play the role of charge buffer to stabilize the negative charge built up on the intermediates in the course of the reaction. The hydrogen bond distances in the intermediate analogues are found to be about 0.2 Angstrom shorter in the product analogues both ex perimentally and theoretically.