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

Hydrolases are a critical component for modern chemical, pharmaceutical, and environmental sciences. Identifying mutations that enhance catalytic efficiency presents a roadblock to design and to discover new hydrolases for broad academic and industrial uses. Here, we report the statistical profiling for rate- perturbing mutant hydrolases with a single amino acid substitution. We constructed an integrated structure-kinetics database for hydrolases, IntEnzyDB, which contains 3907 k(cat)s, 4175 K(M)s, and 2715 Protein Data Bank IDs. IntEnzyDB adopts a relational architecture with a flattened data structure, enabling facile and efficient access to clean and tabulated data for machine learning uses. We conducted statistical analyses on how single amino acids mutations influence the turnover number (i.e., k(cat)) and efficiency (i.e.,k(cat)/K-M), with a particular emphasis on profiling the features for rate-enhancing mutations. The results show that mutation to bulky nonpolar residues with a hydrocarbon chain involves a higher likelihood for rate acceleration than to other types of residues. Linear regression models reveal geometric descriptors of substrate and mutation residues that mediate rate-perturbing outcomes for hydrolases with bulky nonpolar mutations. On the basis of the analyses of the structure-kinetics relationship, we observe that the propensity for rate enhancement is independent of protein sizes. In addition, we observe that distal mutations (i.e., >10 angstrom from the active site) in hydrolases are significantly more prone to induce efficiency neutrality and avoid efficiency deletion but involve similar propensity for rate enhancement. The studies reveal the statistical features for identifying rate-enhancing mutations in hydrolases, which will potentially guide hydrolase discovery in biocatalysis.

Automated summary

Statistical profiling was conducted to identify mutations that enhance catalytic efficiency in hydrolases, revealing that mutations to bulky nonpolar residues are more likely to accelerate reaction rates. The analyses of structure-kinetics relationship showed that the propensity for rate enhancement in hydrolases is independent of protein sizes, and distal mutations have greater potential for inducing efficiency neutrality and avoiding efficiency deletion while still showing similar propensity for rate enhancement.