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Rate-enhancing Single Amino Acid Mutation for Hydrolases: A Statistical Profiling

We reported the statistical profiling for rate-enhancing mutant hydrolases with 
single amino acid substitution. We constructed an integrated structure-kinetics database, 
IntEnzyDB, which contains 3,907 experimentally characterized hydrolase kinetics and 2,715 
hydrolase Protein Data Bank IDs. The hydrolase kinetics data involve 9% rate-enhancing 
mutations. Mutation to nonpolar residues with a hydrocarbon chain shows a stronger preference 
for rate acceleration than to polar or charged residues. To elucidate the structure-kinetics 
relationship for rate-enhancing mutations, we categorized each mutation into one of the three 
spatial shells of hydrolases. We defined the spatial shells by reference to either the active site or
the center-of-mass of the enzyme. In either case, mutations in the first shell (i.e., closest to the 
reference point) appear on average more rate-deleterious than those in the other two shells (i.e., 
~1.0 kcal/mol in ∆∆G‡
). Under the active-site reference, mutations in the third shell (i.e., most 
distal to the active site) exhibit the highest likelihood of rate enhancement. This propensity is 
significant for larger-sized hydrolases. In contrast, under the center-of-mass reference, mutations 
in the second shell (i.e., 33.3th to 66.7th percentile rank of spatial proximity to the center-of-mass 
of the enzyme) show the highest likelihood of rate enhancement. This trend is significant for 
smaller-sized hydrolases. The studies reveal the statistical features for identifying rate-enhancing 
mutations in hydrolases, which will potentially guide hydrolase discovery in biocatalysis.