Abstract
Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5), partially purified from monkey plasma and mouse kidney, has been studied for its substrate specificity and mechanism of action as part of an effort to develop antineoplastic nucleoside analogs that are resistant to enzymic deamination. Steady-state initial rate studies with the enzyme from both sources indicate that uridine is a competitive inhibitor ( K i = 2.8 and 3.4 × 10 −3 M for monkey plasma and mouse kidney deaminase, respectively) with respect to cytidine ( K m = 2.8 × 10 −5 and 1.8 × 10 −4 M for monkey plasma and mouse kidney deaminase, respectively) and ammonium chloride at 2 × 10 −2 M is without inhibitory effect at pH 7.6 or 9.5. The reaction sequence is consistent with a mechanism in which cytidine is the first substrate to interact with the enzyme with the release of uridine as its second product. 1-Methyl-Ψ-isocytidine serves as a substrate; Ψ-isocytidine and 3-deazacytidine do not. Furthermore, Ψ-isocytidine, 2′-deoxy-Ψ-isocytidine and ara-Ψ-isocytosine are potent competitive inhibitors with respect to cytidine in the deaminase reaction ( K i = 7.5 × 10 −5 M, 1.2 × 10 −5 M and 9.2 × 10 −6 M, respectively, for the deaminase from monkey plasma). From substrate specificity and inhibition studies, a mechanism of enzymic deamination of cytosine nucleosides had been proposed which suggests an electrophilic attack by the enzyme at the N-3 position of cytidine which causes electron deficiency at C-4 in the enzyme-substrate complex. Subsequent nucleophilic attack by water at the C-4 position would allow the elimination of ammonia and the dissociation of the enzyme to release uridine as its final product.
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