Kinetic consequences of site-specific mutation of Glu-239----Gln in E. coli aspartate transcarbamylase: comparison with catalytic subunits and Phe-240 mutant enzyme.

The kinetic characteristics of E. coli aspartate transcarbamylase, altered by site-specific mutagenesis of Glu-239----Gln, have been determined by equilibrium isotope-exchange kinetics and compared to the wild-type system. In wild-type enzyme, residue Glu-239 helps to stabilize the T-state structure by multiple bonding interactions with Tyr-165 and Lys-164 across the c1-c4 subunit interface; upon conversion to the R-state, these bonds are re-formed within c-chains. Catalysis of both the [14C]Asp in equilibrium C-Asp and [32P]ATP in equilibrium Pi exchanges by mutant enzyme occurs at rates comparable to those for wild-type enzyme. Saturation with different reactant/product pairs produced kinetic patterns consistent with strongly preferred order binding of carbamyl-P prior to Asp and carbamyl-Asp release before Pi. The kinetics for the Gln-239 mutant enzyme resemble those observed for catalytic subunits (c3), namely a R-state enzyme (Hill coefficient nH = 1.0) and Km (Asp) approximately equal to 6 mM. The Glu-239----Gln mutation appears to destablize both the T- and R-states, whereas the Tyr-240----Phe mutation destablizes only the T-state.