Design and creation of an enzyme having a new function is an important approach in industry. Although, many aftempts has been made to alter the substrate specificity of enzymes, the alteration of the catalytic mechanism of enzymes has not yet been successful. Here, we are presenting the successful alteration of the catalyiic mechanism of an enzyme by amino acid replacement.

We chose T4 phage lysozyme as a model because lysozyme is known as one of the most fnvestigated glycosidases, and there is a large amount of the structural information available. During the investigation ofthe catalyiic mechanism of T4 Iysozyme, we found a mutant enzyme (T26E) which makes the covalent enzyme-substrate adduct artiflcially. From the tertiary structure analysis of this mutant, it was proposed that the covalent linkage was produced because the newly introduced glutamate side chain reacted with the substrate instead of the water molecule existing at the equivalent position in the wild type structure. To confirm the importance of this water molecule, Thr-26 was replaced with other nucleophilic residues, Asp and His. We have found that T26H mutant produces β-anomer as the fmal product, indicating the double displacement mechanism, while the wild type enzyme has a single displacement mechanism which produces α-anomer. This is the flrst example of an alteration of the catalytic mechanism of an enzyme by controlling the water approach to the substrate by amino acid replacement.