Original Paper

JBIC Journal of Biological Inorganic Chemistry

, Volume 17, Issue 7, pp 1123-1134

Open Access This content is freely available online to anyone, anywhere at any time.

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

  • Barbara KrajewskaAffiliated withFaculty of Chemistry, Jagiellonian University Email author 
  • , Rudi van EldikAffiliated withDepartment of Chemistry and Pharmacy, Friedrich Alexander University Erlangen-Nürnberg Email author 
  • , Małgorzata BrindellAffiliated withFaculty of Chemistry, Jagiellonian University

Abstract

Urease, 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), K M and k cat, 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 K M provided the characteristics of the formation of the ES complex, and analyzing k cat, the characteristics of the activation of ES. From the temperature-dependent measurements, the energetic parameters were derived, i.e. thermodynamic ΔH o and ΔS o for ES formation, and kinetic ΔH and ΔS for ES activation, while from the pressure-dependent measurements, the binding ΔV b and activation \( \Updelta V_{\rm cat}^{ \ne } \) 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 abstract

http://static-content.springer.com/image/art%3A10.1007%2Fs00775-012-0926-8/MediaObjects/775_2012_926_Figa_HTML.gif

Keywords

Urease Catalytic mechanism Temperature and pressure dependence Thermodynamic and activation parameters Stopped flow