An Unsual Cys‐Glu‐Lys Catalytic Triad is Responsible for the Catalytic Mechanism of the Nitrilase Superfamily: A QM/MM Study on Nit2

• Published in: Chemphyschem 2021-04, Vol.22 (8), p.796-804
• Main Authors: Silva Teixeira, Carla S., Sousa, Sérgio F., Cerqueira, Nuno M. F. S. A.
• Format: Article
• Language: English
• Subjects: Adducts; catalytic triad; drug discovery; enzyme catalysis; Enzymes; Free energy; Hydrolysis; Nitrilase 2; QM/MM
• ISSN: 1439-4235; 1439-7641
• DOI: 10.1002/cphc.202000751

Nitrilase 2 (Nit2) is a representative member of the nitrilase superfamily that catalyzes the hydrolysis of α‐ketosuccinamate into oxaloacetate. It has been associated with the metabolism of rapidly dividing cells like cancer cells. The catalytic mechanism of Nit2 employs a catalytic triad formed by Cys191, Glu81 and Lys150. The Cys191 and Glu81 play an active role during the catalytic process while the Lys150 is shown to play only a secondary role. The results demonstrate that the catalytic mechanism of Nit2 involves four steps. The nucleophilic attack of Cys191 to the α‐ketosuccinamate, the formation of two tetrahedral enzyme adducts and the hydrolysis of a thioacyl‐enzyme intermediate, from which results the formation of oxaloacetate and enzymatic turnover. The rate limiting step of the catalytic process is the formation of the first tetrahedral intermediate with a calculated activation free energy of 18.4 kcal/mol, which agrees very well with the experimental kcat (17.67 kcal/mol). The catalytic mechanism of Nitrilase 2/ω‐amidase is studied in detail using QM/MM methodologies. The role played by the catalytic triad Cys‐Glu‐Lys is shown to be essential for catalysis. The obtained transition‐state structures can be used to design new inhibitors that can be useful to evaluate the role of the catalytic domain of this enzyme in the progression of different cancer cells.