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

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.