| Abstrakt: |
Isotopic exchange kinetics at chemical equilibrium have been used to identify changes in the regulatory properties of aspartate transcarbamylase (ATCase) caused by site-specific mutation of Tyr240→ Phe (Y240F) in the catalytic chain. With both wild-type and the mutant enzymes, ATP activates both [14C]Asp ƻ N-carbamyl-L-aspartate (C-Asp) and the [32P]carbamyl phosphate (C-P) ƻ Piexchanges. In contrast, with wild-type enzyme, CTP inhibits both exchanges, but with Y240F mutant enzyme CTP inhibits Asp ƻ C-Asp exchange and activates C-P ƻ Piexchange. The bisubstrate analog N-(phosphonacetyl-L-aspartate), PALA, activates Asp ƻ C-Asp at a lower concentration with the Y240F enzyme, but the extent of activation is decreased, relative to wild-type enzyme. PALA activation of C-P ƻ Piobserved with wild-type enzyme disappears completely with the Y240F mutant enzyme. Analysis of perturbations of exchange rates by ATP and CTP were carried out by systematic methods plus computer-based simulations with the ISOBI program. These analyses indicate that (a) ATP increases the rates of association and dissociation for both C-P and Asp, but (b) CTP differentially increases the rate of C-P association to a greater degree than dissociation, but also decreases the rates for Asp association and dissociation in equal propertion. In addition, Arrhenius plots for Y240F ATCase suggest that ATP and CTP act by different mechanisms: ATP increases Vmax(decreases ΔG‡) uniformly at all temperatures, whereas CTP does not alter either Vmax(ΔG‡) or the Arrhenius slope (ΔH‡). |
