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

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