A heavy-atom isotope effect and kinetic investigation of the hydrolysis of semicarbazide by urease from jack bean (Canavalia ensiformis).
A kinetic investigation of the hydrolysis of semicarbazide by urease gives a relatively flat log V/ K versus pH plot between pH 5 and 8. A log V m versus pH plot shows a shift of the optimum V m toward lower pH when compared to urea. These results are explained in terms of the binding of the outer N of the NHNH 2 group in semicarbazide to an active site residue with a relatively low p K a ( approximately 6). Heavy-atom isotope effects for both leaving groups have been determined. For the NHNH 2 side, (15) k obs = 1.0045, whereas for the NH 2 side, (15) k obs = 1.0010. This is evidence that the NHNH 2 group leaves prior to the NH 2 group. Using previously published data from the urease-catalyzed hydrolysis of formamide, the commitment factors for semicarbazide and urea hydrolysis are estimated to be 2.7 and 1.2, respectively. The carbonyl-C isotope effect ( (13) k obs) equals 1.0357, which is consistent with the transition state occurring during either formation or breakdown of the tetrahedral intermediate.
Marlier
John F
JF
Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, California 93407, USA. jmarlier@calpoly.edu
Fogle
Emily J
EJ
Cleland
W W
WW
D032449:Canavalia / Q000201:enzymology; D002247:Carbon Isotopes; D020134:Catalytic Domain; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009532:Nickel / Q000737:chemistry; D009587:Nitrogen Isotopes; D012663:Semicarbazides / Q000737:chemistry / Q000378:metabolism; D014510:Urease / Q000737:chemistry / Q000378:metabolism*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012663:Semicarbazides; C010059:carbamylhydrazine; D009532:Nickel; D014510:Urease