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

Abstract

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 c 1-c 4 subunit interface; upon conversion to the R-state, these bonds are re-formed within c-chains. Catalysis of both the [ 14C] Asp⇌C-Asp and [ 32P]ATP ⇌ P 1 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 P 1. The kinetics for the Gln-239 mutant enzyme resemble those observed for catalytic subunits (c 3), namely a R-state enzyme (Hill coefficient n H = 1.0) and K m ( Asp) ≊ 6 mM . The Glu-239 → Gln mutation appear to destabilize both the T- and R-states, whereas the Tyr-240 → Phe mutation destabilizes only the T-state.