The Mechanism of Action of 5′-Adenylic Acid-activated Threonine Dehydrase

The relationship between the AMP-induced increases in molecular weight and the AMP-promoted decrease in Km for l-threonine for the biodegradative l-threonine dehydrase of Escherichia coli was investigated. The sedimentation velocity in sucrose gradients over a range of 0.07 to 22 µg of dehydrase in the presence of AMP showed little effect of protein concentration (ranging from 7.5 to 8.1 S). In the absence of AMP, the sedimentation velocity increased with protein concentration over the range of 0.61 to 366 µg from 3.7 to 6.4 S. Extrapolation of the line to zero protein concentration indicated a limiting s value of 3.2 S. Oligomers formed at high protein concentration (1 mg ml-1) in the absence of AMP were shown by direct determination in stopped flow assays to have a Km for l-threonine significantly higher than observed in the absence of AMP at much lower protein concentration where monomer would be present. Therefore, oligomer formation alone is not a sufficient condition to cause the allosteric kinetic effect observed with AMP; namely, a decrease in Km for threonine. Following dilution of the concentrated dehydrase in the absence of AMP, an activation was observed upon addition of AMP; the activation was shown to be second order in protein concentration as determined by integral, differential, and fractional time methods. Therefore, the rate limiting step for activation is a dimerization process. These results show that oligomerization is a necessary, although not a sufficient, condition to cause the decrease in Km for threonine elicited by AMP. They also indicate that the sequential isomerization and concerted isomerization models of allosteric behavior are not well suited to the biodegradative threonine dehydrase.