How similar are enzyme active site geometries derived from quantum mechanical theozymes to crystal structures of enzyme-inhibitor complexes? Implications for enzyme design

Abstract

Quantum mechanical optimizations of theoretical enzymes (theozymes), which are predicted catalytic arrays of biological functionalities stabilizing a transition state, have been carried out for a set of nine diverse enzyme active sites. For each enzyme, the theozyme for the rate-determining transition state plus the catalytic groups modeled by side-chain mimics was optimized using B3LYP/6-31G(d) or, in one case, HF/3-21G(d) quantum mechanical calculations. To determine if the theozyme can reproduce the natural evolutionary catalytic geometry, the positions of optimized catalytic atoms, i.e., covalent, partial covalent, or stabilizing interactions with transition state atoms, are compared to the positions of the atoms in the X-ray crystal structure with a bound inhibitor. These structure comparisons are contrasted to computed substrate-active site structures surrounded by the same theozyme residues. The theozyme/transition structure is shown to predict geometries of active sites with an average RMSD of 0.64 A from the crystal structure, while the RMSD for the bound intermediate complexes are significantly higher at 1.42 A. The implications for computational enzyme design are discussed.

Scheme 1.

Bound substrates (substrate–active site complex) and transition state (theozyme) for the DERA-catalyzed aldol reaction.

Scheme 2.

Bound substrates (substrate–active site complex) and transition state (theozyme) for the DmpG-catalyzed retro-aldol reaction.

Scheme 3.

Bound acyl-enzyme intermediate and substrate (water) (substrate–active site complex) and transition state (theozyme) for the BChE-catalyzed hydrolysis of an ester.

Scheme 4.

Bound substrate (substrate–active site complex) and transition state (theozyme) for the Cel5 A-catalyzed hydrolysis of oligosaccaharides.

Scheme 5.

Bound substrate (substrate–active site complex) and transition state (theozyme) in the cytosine deaminase-catalyzed hydrolysis of cytosine to uracil.

Scheme 6.

Bound substrate (substrate–active site complex) and transition state (theozyme) in the carboxypeptidase-catalyzed hydrolysis of a peptide.

Scheme 7.

Bound substrate (substrate–active site complex) and transition state (theozyme) in the aspartic proteinase-catalyzed hydrolysis of a peptide.

Scheme 8.

Bound substrate (substrate–active site complex) and transition state (theozyme) in the Tdp1-catalyzed hydrolysis of a phosphodiester.

Scheme 9.

Bound reactant (substrate–active site complex) and transition state (theozyme) in the conversion of chorismate to prephenate in chorismate mutase.

Figure 1.

Optimized structures for butyrylcholinesterase and catalytic atom (sphere representation) overlays with the crystal structure (wire-frame representation) for (A) ester substrate bound, (B) TS for nucleophilic attack of the serine side chain to carbonyl carbon of ester, and (C) TS for histidine hydrogen-bond migration from the serine side-chain oxygen to the ester oxygen of the substrate.

Figure 2.

Summary of RMSD (Å) for optimized theozymes and active site–substrate complexes compared to the corresponding crystal structure.