Abstract
The urease-catalyzed hydrolysis of hydroxyurea is known to exhibit biphasic kinetics, showing a rapid burst phase followed by a slow plateau phase. Kinetic isotope effects for both phases of this reaction were measured at pH 6.0 and 25 °C. The observed nitrogen isotope effects for the ammonia leaving group [(15)(V/K)(NH(3))] were 1.0016 ± 0.0005 during the burst phase and 1.0019 ± 0.0007 during the plateau phase, while those for the hydroxylamine leaving group [(15)(V/K)(NH(2)OH)] were 1.0013 ± 0.0005 for the burst phase and 1.0022 ± 0.0003 for the plateau phase. These isotope effects are consistent with a rate-determining step that occurs prior to breaking either of the two possible C-N bonds. The observed carbonyl carbon isotope effects [(13)(V/K)] were 1.0135 ± 0.0003 during the burst phase and 1.0178 ± 0.0003 during the plateau phase. The similarity of the magnitude of the carbon isotope effects argues for formation of a common intermediate during both phases.