Inhibition of Escherichia coli cytidine deaminase by a phosphapyrimidine nucleoside.
The nature of the interaction between Escherichia coli cytidine deaminase and the phosphapyrimidine nucleoside 1 has been studied kinetically and spectrophotometrically. Compound 1 was designed as a transition-state analog, and is a potent, slow-binding inhibitor of cytidine deaminase (Ashley, G. W., and Bartlett, P. A. (1982) Biochem. Biophys. Res. Commun. 108, 1467-1474). We present evidence that the binding of 1 is reversible, with no covalent linkage between the enzyme and 1. At pH 6, the rate of recovery of enzyme activity from dissociation of the E X I complex is strongly dependent on the concentration of E X I, indicating that the inhibitor dissociates reversibly. UV difference spectroscopy reveals that the chromophore of 1 is unaltered on binding to the enzyme, thus eliminating the possibility of reversible, covalent modification of the enzyme. For the binding of the active beta-anomers of 1 to cytidine deaminase, the following kinetic parameters were determined at pH 6: kon = 8300 M-1 S-1, koff = 7.8 X 10(-6) S-1, Ki = 0.9 nM. We were also able to observe and characterize time-dependent inhibition of E. coli cytidine deaminase by tetrahydrouridine, 3. This interaction involves involves initial formation of a loose complex (KD = 1.2 microM), followed by isomerization in a slow step to give a more tightly bound complex (Ki = 0.24 microM) with forward and reverse rate constants kf = 3.81 min-1 and kr = 0.95 min-1, respectively.
D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D009700:Nucleoside Deaminases / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D011741:Pyrimidine Nucleosides / Q000138:chemical synthesis / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D013767:Tetrahydrouridine / Q000494:pharmacology
D007202:Indicators and Reagents; D011741:Pyrimidine Nucleosides; C043412:phosphapyrimidine ribonucleoside 1; D013767:Tetrahydrouridine; D009700:Nucleoside Deaminases; D003564:Cytidine Deaminase