Urease, a Ni-containing metalloenzyme, fea- tures 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 pres- sures. 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 parame- ters in a noninteracting buffer using a stopped-flow tech- nique in the temperature range 15–35 ° C and in the pressure range 5–132 MPa, the pressure-dependent mea- surements being the first of their kind performed for urease. While temperature enhanced the activity of urease, pres- sure 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 D H o and D S o for ES formation, and kinetic D H

and D S

for ES activation, while from the pressure- dependent measurements, the binding D V b and activation D V 6 ¼ cat 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.