Ab initio determination of the structure of the active site of a metalloenzyme: Metal substitution in phosphotriesterase using density functional methods

A number of previous studies have determined that a metalloenzyme active si te is inherently determined by the ligands and metal and only weakly pertur bed by the surrounding protein environment. This conclusion is now being te sted for several families of bimetallic enzymes. Metal substitution is exam ined for phosphotriesterase (PTE) with all possible substitutions of zinc a nd cadmium in the two sites. A new density functional theory (DFT) function al is used in this study which has yielded accurate structures and thermoch emistry for molecules made up of first and second row atoms. Comparisons be tween gradient-optimized Hartree-Fock and DFT structures are in good agreem ent, suggesting this functional is useful in studying transition metal enzy me active sites. Good agreement between the X-ray and in vacuo optimized st ructures is also obtained for the Zn-Zn and Cd-Cd enzymes. This supports th e analysis of the PTE active site as an inherent complex and suggests that accurate structures can be obtained for other metal substitutions for which no experimental structures are available. A unique zinc/cadmium hybrid was observed experimentally, and the structure of this active site is theoreti cally predicted. The polarization of bound water at this very polar active site is very large, suggesting it is the reactive nucleophile. (C) 1999 Joh n Wiley & Sons, Inc.