Multiple isotope effect study of the hydrolysis of formamide by urease from jack bean (Canavalia ensiformis)

Multiple kinetic isotope effects have been measured for the urease-catalyzed hydrolysis of formamide at pH 6.0 and 25 degrees C. These kinetic isotope effects include the carbonyl-C ((13)k = 1.0241 +/- 0.0009), the carbonyl-O ((18)k = 0.9960 +/- 0.0009), the formyl-H ((D)k = 0.95 +/- 0.01), the leaving-N ((15)k= 1.0327 +/- 0.0006), and the nucleophile-O ((18)k = 0.9778 +/- 0.0005). In addition, the enzyme does not catalyze the exchange of oxygen from the solvent into the carbonyl-O of formamide or the product, formate ion. The isotope effects are consistent with the rate-determining collapse of the tetrahedral intermediate (i.e., C-N bond cleavage). The pH optimum for formamide is at pH 5.3, whereas for urea, it is near 8.0. This is best accommodated by the mechanism proposed by Hausinger and Karplus, in which an active site cysteine binds to the nonleaving nitrogen in urea. For urea, the preference is for the anionic form of the sulfhydryl; for formamide, the neutral form is preferred, leading to the lower pH optimum.