title: Site-specific mutation of Tyr240----Phe in the catalytic chain of Escherichia coli aspartate transcarbamylase. Consequences for kinetic mechanism.
abstract: In the catalytic chain of Escherichia coli aspartate transcarbamylase, Tyr240 helps stabilize the T-state conformation by an intrachain hydrogen bond to Asp271. Changes in kinetic characteristics of ATCase that result from disruption of this bond by site-specific mutation of Tyr240----Phe have been investigated by isotopic exchanges at chemical equilibrium. The Tyr240----Phe (Y240F) mutation caused the rate of the [32P] carbamyl phosphate (C-P) in equilibrium Pi exchange to decrease by 2-8-fold, without altering the [14C]Asp in equilibrium N-carbamyl-L-aspartate (C-Asp) rate. The mutation also caused the S0.5 and Hill nH values to decrease in virtually every substrate saturation experiment. Upon increasing the concentrations of the C-P,Pi or C-P,C-Asp reactant-product pairs, inhibition effects observed with the C-P in equilibrium Pi exchange for wild-type enzyme were not apparent with the Y240F mutant enzyme. In contrast, upon increasing the concentrations of the Asp,C-Asp and Asp,Pi pairs, inhibition effects on C-P in equilibrium Pi observed with wild-type enzyme became stronger with the Y240F mutant enzyme. These data indicate that the Tyr240----Phe mutation alters the kinetic mechanism in two different ways: on the reactant side, C-P binding prior to Asp shifts from preferred to compulsory order, and, on the product side, C-Asp and Pi release changes from preferred to nearly random order. These conclusions were also confirmed on a quantitative basis by computer simulations and fitting of the data, which also produced an optimal set of rate constants for the Y240F enzyme. The Arrhenius plot for wild-type holoenzyme was biphasic, but those for catalytic subunits and Y240F enzyme were linear (monophasic). Taken together, the data indicate that the Tyr240----Phe mutation destabilizes the T-state and shifts the equilibrium for the T-R allosteric transition toward the R-state by increasing the rate of T----R conversion.
authors.affiliation: Department of Molecular & Cell Biology, P.M. Althouse Laboratory, Pennsylvania State University, University Park 16802.
authors.forename: Y
authors.initials: Y
authors.lastname: Hsuanyu
authors.forename: F C
authors.initials: FC
authors.lastname: Wedler
authors.forename: E R
authors.initials: ER
authors.lastname: Kantrowitz
authors.forename: S A
authors.initials: SA
authors.lastname: Middleton
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism / Q000031:analogs & derivatives; D002221:Carbamyl Phosphate / Q000378:metabolism; D002384:Catalysis; D003198:Computer Simulation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D010649:Phenylalanine; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014443:Tyrosine
chemical_list: D010710:Phosphates; D001224:Aspartic Acid; D014443:Tyrosine; D010649:Phenylalanine; D002221:Carbamyl Phosphate; D001221:Aspartate Carbamoyltransferase; C006893:ureidosuccinic acid