2 to N-hydroxy-cytidine 4 catalyzed by cytidine deaminase. Pathway A involves direct conversion of 2 to 4 using NH 2 OH as a nucleophile. Pathway B involves initial hydrolysis of 2 to uridine 3 which is subsequently transformed to 4 through condensation with NH2OH. Pathway B is the dominant pathway when using the wild-type enzyme, leading to an equilibrium distribution of 3 and 4 that is dependent on NH2OH concentration. b) The conversion of cytidine 2 (1mM) and uridine 3 (1mM) to N-hydroxy-cytidine 4 by CD (5 μM) in the presence of 1% NH2OH (pH 7) is monitored by increasing absorbance at 310 nm. Similar final concentrations of N-hydroxy-cytidine are formed using either 2 (red) or 3 (blue) as a starting material, or in reactions starting from N-hydroxy-cytidine 4 (green). c) The conversion of cytidine 2 (750 mM) by CD (25 μM) to uridine 3 to N-hydroxy-cytidine 4 is monitored by HPLC analysis. The time course of the reaction indicates CD operates via pathway B.