A Heavy-Atom Isotope Effect and Kinetic Investigation of the Hydrolysis of Semicarbazide by Urease from Jack Bean (Canavalia ensiformis)

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• 作者：John F. Marlier ; Emily J. Fogle ; W. W. Cleland
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：October 21, 2008
• 年：2008
• 卷：47
• 期：42
• 页码：11158-11163
• 全文大小：148K
• 年卷期：v.47,no.42(October 21, 2008)
• ISSN：1520-4995

文摘

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 NHNH₂ group in semicarbazide to an active site residue with a relatively low pK_a (∼6). Heavy-atom isotope effects for both leaving groups have been determined. For the NHNH₂ side, ¹⁵k_obs = 1.0045, whereas for the NH₂ side, ¹⁵k_obs = 1.0010. This is evidence that the NHNH₂ group leaves prior to the NH₂ 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 (¹³k_obs) equals 1.0357, which is consistent with the transition state occurring during either formation or breakdown of the tetrahedral intermediate.