Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Abstract

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [ 14C]Asp C-Asp exchange is much faster than [ 32P]carbamyl phosphate P i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E a (>15 °C) = 6.3 kcal/ mole and E a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E a ≅ 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.