Role of aspartokinase substrates in the dehydrogenase activity of the aspartokinase-homoserine dehydrogenase complex

Broussard, Larry A.; Cunningham, Glenn N.; Starnes, Willis L.; Shive, William

doi:10.1016/s0006-291x(72)80099-8

Cited by 6 publications

(1 citation statement)

References 5 publications

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“…As the salt concentration was increased to 0.8 M, Tmjn decreased from 0.17 to 0.13 s. The Hill coefficient decreased from 2.7 to 2.15 over the same range of salt concentrations, but this decrease is not significant because of the error associated with values of «h-Similar conclusions are reached with studies of MgATP. Because of the multiple effect of MgATP on the HSD activity (Patte et al, 1966;Cunningham et al, 1968;Broussard et al, 1972;Ogilvie et al, 1975), the effect of its addition to stopped-flow solutions was examined. The decrease in Tmin upon addition of 0.25 MgATP was small but significant, but no significant change was observed for the Hill coefficients (Figure 4).…”

Section: Discussionmentioning

confidence: 99%

Threonine inhibition of the aspartokinase-homoserine dehydrogenase I of Escherichia coli. Stopped-flow kinetics and the cooperativity of inhibition of the homoserine dehydrogenase activity

Bearer¹,

Neet²

1978

Biochemistry

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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, k i, 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, rmi", and the threonine concentration at half the maximal rate of transition, AT, were obtained from analysis of doublereciprocal plots of k i vs. threonine concentration. rmjn was substantially decreased with increasing temperature. Other changes in experimental conditions resulted in smaller decreases in rmin. Kinetic Hill coefficients obtained from standardThe presence of two distinct, separate sites of threonine binding to aspartokinase-homoserine dehydrogenase I (AK-HSD)* 1 of E. coli (Bearer & Neet, 1978) has raised the question of the role of each in the inhibition of homoserine dehydrogenase activity, and the mechanism by which inhibition occurs. Thus, a study of the initial and steady-state kinetics of the HSD inhibition was undertaken. The characteristics of the steady-state inhibition of HSD by threonine vary according to direction and conditions of assay. Under forward assay conditions (aspartic /3-semialdehyde to homoserine) threonine inhibition is noncompetitive vs. aspartic /3-semialdehyde (Patte et al., 1963). When studied at high pH (8.5) and low potassium (5 mM), no cooperativity is observed (Truffa-Bachi et al., 1974). At lower pH and in the presence of 0.

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Section: Discussionmentioning

confidence: 99%