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

Abstract

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.