A molecular dynamics exploration of the catalytic mechanism of yeast cytosine deaminase

Yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, is investigated by a series of molecular dynamics simulations in its free form and complexed with its reactant (cytosine), product (uracil), several reaction intermediates, and an intermediate analogue. Quantum chemical calculations, used to construct a model for the catalytic Zn ion with its ligands (two cysteines, a histidine, and one water) show, by comparison with crystal structure data, that the cysteines are deprotonated and the histidine is monoprotonated. The simulations suggest that Glu64 plays a critical role in the catalysis by yCD. The rotation of the Glu64 side-chain carboxyl group that can be protonated or deprotonated permits it to act as a proton shuttle between the Zn-bound water and cytosine and subsequent reaction intermediates. Free energy methods are used to obtain the barriers for these rotations, and they are sufficiently small to permit rotation on a geometry to favor nucleophilic nanosecond time scale. In the course of the reaction, cytosine reorients to a attack by a Zn-bound hydroxide. A stable position for a reaction product, ammonia, was located in the active site, and the free energy of exchange with a water molecule was evaluated. The simulations also reveal small motions of the C-terminus and the loop that contains Phe114 that may be important for reactant binding and product release.