Mori, S.; Niide, T.; Toya, Y.; Shimizu, H.

Identifying amino acid residues that are critical for the catalytic function of enzymes is essential for elucidating reaction mechanisms, facilitating drug discovery, and advancing protein engineering. However, experimentally and computationally distinguishing residues that maintain structural integrity from those directly involved in enzymatic function remains a major challenge. In this study, we developed a methodology to identify amino acid residues that influence substrate specificity in enzymes with homologous structures. We framed the sequence comparison as a classification problem, treating each residue as a feature, thereby enabling the rapid and objective identification of key residues responsible for functional differences. To validate the proposed method, we applied it to three enzyme pairs - trypsin/chymotrypsin, adenylyl cyclase/guanylyl cyclase, and lactate dehydrogenase (LDH)/malate dehydrogenase (MDH). The results confirmed the accurate prediction of previously identified specificity-determining residues. Furthermore, we conducted experiments on the LDH/MDH pair and successfully introduced mutations into key residues to alter substrate specificity, enabling LDH to utilize oxaloacetate while maintaining its expression levels. These findings demonstrate the potential of this method for efficiently identifying residues that govern substrate specificity. We have further developed this approach into a practical tool, the EZSCAN: Enzyme Substrate-specificity and Conservation Analysis Navigator (https://ezscan.pe-tools.com/), which enables rapid identification of amino acid residues critical for enzyme function.