Product release mechanism and the complete enzyme catalysis cycle in yeast cytosine deaminase (yCD): A computational study

- Five plausible channels for the product (uracil) release step are detected.
- The thermodynamic and dynamic properties for the two most favorable channels are obtained.
- The key residues that are responsible for the barrier of product release have been found.
- The full cycle of the enzymatic catalysis is determined.

Yeast cytosine deaminase (yCD) is critical in gene-directed enzyme prodrug therapy as it catalyzes the hydrolytic deamination of cytosine. The product (uracil) release process is considered as rate-limiting in the whole enzymatic catalysis and includes the cleavage of the uracil-metal bond and the delivery of free uracil out of the reactive site. Herein extensive combined random acceleration molecular dynamics (RAMD) and molecular dynamics (MD) simulations coupled with the umbrella sampling technique have been performed to study the product transport mechanism. Five channels have been identified, and the thermodynamic and dynamic characterizations for the two most favorable channels have been determined and analyzed. The free energy barrier for the most beneficial pathway is about 13 kcal/mol and mainly results from the cleavage of hydrogen bonds between the ligand uracil and surrounding residues Asn51, Glu64, and Asp155. The conjugated rings of Phe114 and Trp152 play gating and guiding roles in the product delivery via π ⋯ π van der Waals interactions with the product. Finally, the full cycle of the enzymatic catalysis has been determined, making the whole process computationally more precise.

The kinetic model for the yCD catalytic process.193