Substrate assistance in the mechanism of family 18 chitinases: Theoretical studies of potential intermediates and inhibitors

Based on first principles and molecular mechanics calculations, we conclude that the mechanism of hevamine (a family 18 chitinase) involves an oxazoline ion intermediate stabilized by the neighboring C2' acetamido group. In this intermediate, the acetamido carbonyl oxygen atom forms a covalent bond to C1' of N-acetyl-glucosamine and has a transferred positive charge from the pyranose ring onto the acetamido nitrogen atom, leading to an anchimeric stabilization of 38.1 kcal/mol when docked with hevamine. This double displacement mechanism involving an oxazoline intermediate distinguishes the family 18 chitinase (which have one acidic residue near the active site) from family 19 chitinase and from hen egg-white lysozyme, which have two acidic residues near the active site. The structural and electronic properties of the oxazoline intermediate are similar to the known chitinase inhibitor allosamidin, suggesting that allosamidins act as transition state analogs of an oxazoline intermediate. Structural and electronic features of the oxazoline ion likely to be important in the design of new chitinase inhibitors are discussed.