Effect of thermodynamic nonideality in kinetic studies: evidence for reversible unfolding of urease during urea hydrolysis.

A combination of enzyme kinetic studies and active enzyme gel chromatography on Sepharose CL-6B was used to explore conformational changes of the enzyme urease as it catalyzes the hydrolysis of urea in 0.7 M phosphate buffer, pH 7.0, at 20 degrees C. It is shown that elucidation of this system is only possible by studying the effects of inert space-filling macromolecules (ovalbumin and bovine serum albumin) on enzymatic behavior. The resulting increases in reaction velocity are interpreted in terms of composition-dependent activity coefficients assessed on a statistical mechanical basis of excluded volume. The results are first considered in terms of two extreme models; one involving a volume change on the isomerization of the enzyme-substrate complex to its activated state, and the other an isomeric expansion of the enzyme-substrate complex to an inactive form. Although both extreme models provide satisfactory descriptions of the kinetic results, they lead to unrealistic values for the radii of the various states of the enzyme-substrate complex. It is concluded, therefore, that the two isomeric transitions act conjointly, a result in conformity with the previously postulated conformational change associated with formation of the activated enzyme-substrate complex [L. W. Nichol, M. J. Sculley, L. D. Ward, and D. J. Winzor (1983) Arch. Biochem. Biophys. 222, 574-581], and also with the well-established action of the substrate, urea, as an unfolding agent of proteins.