L-aspartate association contributes to rate limitation and induction of the T-->R transition in Escherichia coli aspartate transcarbamoylase. Equilibrium exchanges and kinetic isotope effects with a Vmax-enhanced mutant, Asp-236-->Ala.
Equilibrium isotope exchange kinetics (EIEK) and kinetic isotope effects have been used to determine the mechanistic basis for the altered kinetic characteristics of a mutant version of Escherichia coli aspartate transcarbamylase in which Asp-236 of the catalytic chain is replaced by alanine (Asp-236-->Ala). The [14C]Asp<--> N-carbamyl-L-aspartate (CAsp) and [14C]CP<-->CAsp exchange rates, observed as a function of various reactant-product pairs, exhibited dramatic increases in maximal rates, along with decreases in substrate half-saturation values and cooperativity. The carbon kinetic isotope effect, 13C versus 12C at the carbonyl group of carbamoyl phosphate, for the Asp-236-->Ala enzyme decreased toward unity as [Asp] increased, as observed for the wild-type enzyme. Both the kinetic isotope effects and EIEK results indicate that the Asp-236-->Ala enzyme operates by the same ordered kinetic mechanism as the wild-type enzyme. Although activation effects by ATP and N-phosphonacetyl-L-aspartate are lost, inhibition by CTP was apparent in equilibrium exchanges. Simulation of the EIEK data indicated that the best fit to the observed changes in saturation curves was obtained by preferentially increasing the rate of the T-->R transition, kappa T-->R, thereby destabilizing the T-state and increasing the equilbrium constant for the T<-->R transition. A multistep model for Asp bindng to aspartate transcarbamoylase is proposed, in which Asp induces the initial conformational changes that in turn trigger the T-->R transition, followed by stepwise filling of the remaining active sites.
Wedler
F C
FC
Department of Biochemistry and Molecular Biology, Althouse Laboratory, Pennsylvania State University, University Park, USA.
Ley
B W
BW
Lee
B H
BH
O'Leary
M H
MH
Kantrowitz
E R
ER
D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007554:Isotopes; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D017354:Point Mutation
D007554:Isotopes; D008024:Ligands; D001224:Aspartic Acid; D001221:Aspartate Carbamoyltransferase; D000409:Alanine