Independent Modification of the Binding Sites for Adenosine Diphosphate and L-Threonine in Threonine Dehydratase

Abstract

Abstract The reactions between l-threonine dehydratase from Clostridium tetanomorphum and N-ethylmaleimide, tetranitromethane, iodine, fluorodinitrobenzene, or diazobenzene sulfonic acid were studied at 2° in order to determine the effects of chemical modification on certain kinetic parameters. At times from 0 to 1 hour after treatment, samples were taken for the determination of Michaelis constant (Km) for l-threonine and the maximum velocity of dehydration (Vmax). The data revealed that N-ethylmaleimide at pH 6.5 and tetranitromethane at pH 6.3 were able to alter catalytic ability as determined by a 10-fold decrease in Vmax. The Km for threonine was not appreciably affected under these conditions. The substrate analogue, allothreonine, was protective against the changes in Vmax. Iodination at pH 7.6 or pH 6.3 modified the enzyme so that the Km for threonine increased up to 4-fold, but Vmax was unaffected. At pH 6.3, the presence of ADP during iodination prevented the increase in Km but allothreonine was without effect. Tetranitromethane at pH 8.3 or fluorodinitrobenzene and diazobenzene sulfonic acid at pH 7.6 modified the enzyme such that the Km for threonine increased 3- to 5-fold and Vmax decreased to 10% the original value. In most cases, ADP could protect against large increases in Km but not against changes in Vmax, whereas allothreonine could prevent decreases in Vmax but was largely ineffective against increases in Km. Protection by ADP or allothreonine was generally more effective at pH 6.3 to 6.5 than at pH 7.6 or higher. The extent of change in Km upon chemical modification was approximately the same as that seen with native dehydratase assayed in the absence and presence of ADP. In both cases, Km increased to near 70 mm from the 20 mm value obtained for the native ADP-activated enzyme. Concomitant with the increase in Km promoted by iodination or dinitrophenylation, Ka for ADP increased dramatically. The results presented provide a correlation between modifications which are protected by ADP and changes in the Michaelis constant for l-threonine, as well as between modifications which are protected by allothreonine and decreases in Vmax for dehydration. These kinetic parameters seem to indicate that sites specifically protected by allothreonine or ADP can be modified through appropriate choice of reagent and conditions. In these sites can be equated with the binding sites for substrate and ADP, then it would appear that important differences exist in the composition or degree of exposure of certain reactive amino acids associated with each site.