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15 N Kinetic Isotope Effects on Uncatalyzed and Enzymatic Deamination of Cytidine

2001; American Chemical Society; Volume: 41; Issue: 1 Linguagem: Inglês

10.1021/bi011410i

1943-295X

Mark J. Snider, Laurie A. Reinhardt, Richard Wolfenden, W. W. Cleland,

Pancreatic function and diabetes

15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary 15(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H2O, pH 7.3), 1.0123 (in H2O, pH 4.2), and 1.0086 (in D2O, pD 7.3). Increasing solvent D2O content has no substantial effect on kcat but enhances kcat/Km, with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced 15N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H2O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH2 group, and represents the intrinsic isotope effect on C−N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed 15N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary 15N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 °C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C−N bond breaking is greater for the spontaneous reaction.