Temperature- and pressure-dependent stopped-flow kinetic studies of jack bean urease. Implications for the catalytic mechanismReport as inadecuate

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JBIC Journal of Biological Inorganic Chemistry

, Volume 17, Issue 7, pp 1123–1134

First Online: 14 August 2012Received: 30 April 2012Accepted: 14 July 2012

AbstractUrease, a Ni-containing metalloenzyme, features an activity that has profound medical and agricultural implications. The mechanism of this activity, however, has not been as yet thoroughly established. Accordingly, to improve its understanding, in this study we analyzed the steady-state kinetic parameters of the enzyme jack bean, KM and kcat, measured at different temperatures and pressures. Such an analysis is useful as it provides information on the molecular nature of the intermediate and transition states of the catalytic reaction. We measured the parameters in a noninteracting buffer using a stopped-flow technique in the temperature range 15–35 °C and in the pressure range 5–132 MPa, the pressure-dependent measurements being the first of their kind performed for urease. While temperature enhanced the activity of urease, pressure inhibited the enzyme; the inhibition was biphasic. Analyzing KM provided the characteristics of the formation of the ES complex, and analyzing kcat, the characteristics of the activation of ES. From the temperature-dependent measurements, the energetic parameters were derived, i.e. thermodynamic ΔH and ΔS for ES formation, and kinetic ΔH and ΔS for ES activation, while from the pressure-dependent measurements, the binding ΔVb and activation \ \Updelta V { m cat}^{ e } \ volumes were determined. The thermodynamic and activation parameters obtained are discussed in terms of the current proposals for the mechanism of the urease reaction, and they are found to support the mechanism proposed by Benini et al. Structure 7:205–216; 1999, in which the Ni–Ni bridging hydroxide—not the terminal hydroxide—is the nucleophile in the catalytic reaction.

Graphical abstractOpen image in new windowKeywordsUrease Catalytic mechanism Temperature and pressure dependence Thermodynamic and activation parameters Stopped flow  Download fulltext PDF

Author: Barbara Krajewska - Rudi van Eldik - Małgorzata Brindell

Source: https://link.springer.com/

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