Modeling of reaction steps relevant to deoxyuridylate (dump) enzymatic methylation and thymidylate synthase mechanism-based inhibition

Theoretical quantum mechanical ab initio Hartree-Fock calculations on molecular systems, modeling processes related to the specificity of thymidylate synthase inactivation are reported. We considered several steps of the methylation of the substrate dUMP and 4- or 5-mono- and 4,5-bisubstituted dUMP analogs, as well. The following reactions were modeled: the cysteine residue (Cys¹⁹⁸ in the L. casei enzyme) nucleophilic attack on the substrate and the substrate C(5)-H proton abstraction. The substrate was modeled by the 1-methyluracil molecule and its structural analogs. The cysteine Cys¹⁹⁸ residue was modeled by the methylmer-captane molecule. The substrate-enzyme binary complex was modeled by the 1-methy 1–5, 6- dihydro-6-thiomethyl-uracil (PI) molecule. The present theoretical calculations suggest that the cysteine nucleophilic attack on the substrate may result in the SH-group addition to the pyrimidine C(5)=C(6) bond in the course of a weakly exothermic reaction. The formerly presumed enolate carbanion appeared to be weakly stable or unstable and it can readily split into the thiol and pyrimidine residues. The s²-thio- (P2) and s²⁴-dithio- (P3) substrate analogs should form stable thiolate anions after cysteine residue attachment to the C(6) position of the pyrimidine ring. Studies of the deformed PI molecule interacting with a water molecule bound to the pyrimidine C(4)=0 carbonyl residue allow a suggestion that this water molecule may be directly involved in the C(5)-H proton abstraction and may serve as a proton transmitter between the substrate and the proton acceptor residue, possibly located on the cofactor N¹⁰-nitrogen. Interaction of the pyrimidine C(4)=0 group, or its modification, with the N^5,10-methylenetetrahydrofolate N(10) nitrogen atom is suggested as an additional factor influencing the inhibition process.