Influence of nucleotide effectors on the kinetics of the quaternary structure transition of allosteric aspartate transcarbamylase.
We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.
Tsuruta
Hiro
H
Stanford Synchrotron Radiation Laboratory, SLAC, MS 69, 2575 Sand Hill Rd, Menlo Park, CA 94025-7015, USA. tsuruta@slac.stanford.edu
Kihara
Hiroshi
H
Sano
Takayuki
T
Amemiya
Yoshiyuki
Y
Vachette
Patrice
P
D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D020836:Protein Structure, Quaternary
D009711:Nucleotides; D001221:Aspartate Carbamoyltransferase