THREONINE INHIBITION OF ASPARTOKINASE-HOMOSERINE DEHYDROGENASE-I OF ESCHERICHIA-COLI - STOPPED-FLOW KINETICS AND COOPERATIVITY OF INHIBITION OF HOMOSERINE DEHYDROGENASE-ACTIVITY

Threonine interactions with the threonine-sensitive aspartokinase-homoserine dehydrogenase I (AK-HSD) from Escherichia coli K12 have been investigated by studying the relaxation times from the uninhibited to the inhibited form of the enzyme and the kinetic inhibition of the steady state activity. Comparison of changes in the relaxation time, changes in the cooperativity of the inhibition, and changes in binding cooperativity have been made under a range of comparable conditions. The progress curve lag in threonine inhibition of the HSD activity was studied by stopped-flow rapid kinetic techniques as a function of pH, temperature, ionic strength, and ATP concentration. The protein concentration independence and the activation energy of the apparent first-order rate constant for the lag, k1, demonstrated its identity to the R to T isomerization previously studied. Observed relaxation times ranged from 10 ms to 1.5 s. Limiting values of the relaxation time, Tmin, and the threonine concentration at half the maximal rate of transition, Kk, were obtained from analysis of doublereciprocal plots of k1 vs. threonine concentration. Tmin was substantially decreased with increasing temperature. Other changes in experimental conditions resulted in smaller decreases in Tmin. Kinetic Hill coefficients obtained from standard spectrophotometric procedures were unaffected by changes in temperature, remaining constant at about 2.2. A small increase of the Hill coefficient with increasing pH and a small decrease on addition of 0.8 M choline chloride were observed. Lack of correlation of changes in Tmin and nh ruled out a kinetic contribution of the lag of inhibition to the cooperativity observed for threonine inhibition of the HSD activity. A comparison of K1, the threonine inhibition constant, and Kk, the threonine concentration at half the maximal rate of transition, revealed that no correlation existed between these two parameters, and that the value of K1 is an order of magnitude lower than that of Kk. Values of the Hill c efficient and dissociation constant for binding to the high affinity site under various conditions were equivalent to the corresponding values for inhibition of the HSD steady-state kinetics. The high affinity sites are proposed to be responsible for HSD inhibition and the kinetic cooperativity for this activity is solely a reflection of the binding cooperativity. We conclude that the cooperative inhibition of HSD activity by threonine is best described by rapid equilibrium binding of threonine to the high affinity site with subsequent site-site interactions. © 1978, American Chemical Society. All rights reserved.