Abstract

In the post-genomic era, genome sequencing has rapidly advanced, but our ability to assign functions to genes—especially enzymes—lags behind. Many enzymes remain uncharacterized or misannotated, limiting our understanding of metabolism and biology. Enzymes drive essential chemical reactions in cells, but identifying their specific roles, including substrates, products, and biological context, remains a major challenge. While bioinformatics tools can predict functions based on sequence similarity, they often fail for enzymes without known homologs. Structural and machine learning approaches also struggle with the complexity of enzyme chemistry. Experimental techniques, particularly high-resolution mass spectrometry, have become vital for discovering new enzymatic activities by directly detecting substrates, intermediates, and products. Accurate enzyme annotation is crucial not only for advancing basic science but also for enabling progress in drug development, biotechnology, and synthetic biology.