Deriving Chemically Essential Interactions Based on Active Site Alignments and Quantum Chemical Calculations: A Case Study on Glycoside Hydrolases

Abstract

We here use an approach of active site alignment and clustering of many evolutionarily distant enzymes catalyzing alike reactions to identify conserved residues/interactions that may play key chemical roles in catalysis. Then density functional theory (DFT) calculations on cluster models are used to investigate the chemical essentialness of such residues/interactions and its mechanistic basis. We apply this approach to 130 glycoside hydrolases (GHs) of the (βα)8-barrel fold. These enzymes adopt either a classical retaining mechanism or a substrate-assisted intramolecular nucleophilic attack mechanism, both in need of a general acid/general base residue for catalysis. On the basis of the multiple active site alignments, the enzyme active sites can be clustered into six categories. The conserved or convergently evolved hydrogen bond/salt bridge involving the general acid/general base in different categories suggests the importance of this interaction. DFT calculations indicate that its presence may reduce the energetic barrier by as large as 17–20 kcal mol–1. The mechanistic explanation for this large effect is that a proton transfer from the general acid to the leaving group takes place before the nucleophile attacks the transition state. The large energetic effect suggests that this interaction should be considered as chemically essential, although it is realized with varied residue types in different GH categories. In addition, for the substrate-assisted mechanism, an interaction between the substrate nucleophile group and a tyrosine is found to have been convergently evolved in enzymes of two different categories. This interaction does not seem to have favorable effects on the energetic barrier. Instead, it might contribute to reducing the activation entropy. In summary, active site alignment of distant enzymes combined with quantum mechanical calculation may comprise a powerful approach to obtain new insights into enzyme catalysis.