Role of glutamate 64 in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both the binding and the chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.

Product release is rate-limiting in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

Yeast cytosine deaminase (yCD), a zinc metalloenzyme, catalyzes the hydrolytic deamination of cytosine to uracil. The enzyme is of great biomedical interest because it also catalyzes the deamination of the prodrug 5-fluorocytosine (5FC) to form the anticancer drug 5-fluorouracil (5FU). yCD/5FC is one of the most widely used enzyme/prodrug combinations for gene-directed enzyme prodrug therapy for the treatment of cancers. A pH indicator assay has been developed for the measurement of the steady-state kinetic parameters for the deamination reaction. Transient kinetic studies have shown that the product release is a rate-limiting step in the activation of the prodrug 5FC by yCD. The rate constant of the chemical step for the forward reaction (250 s(-)(1)) is approximately 8 times that of the product release (31 s(-)(1)) and approximately 15 times k(cat) (17 s(-)(1)). The transient kinetic results are consistent with those of the steady-state kinetic analysis in the sense that the k(cat) and K(m) values calculated from the rate constants determined by the transient kinetic analysis are in close agreement with those measured by the steady-state kinetic analysis. NMR experiments have demonstrated that free 5FU is in slow exchange with its complex with yCD but has a low affinity for yCD. The transient kinetic and NMR results together suggest that the release of 5FU is rate-limiting in the activation of the prodrug 5FC by yCD and may involve multiple steps.