Conformation of cross-linked aspartate transcarbamoylase.
We have previously shown that aspartate transcarbamylase loses its substrate cooperativity after modification with a cross-linking reagent. Depending on the presence or absence of substrate analogues during cross-linking, the derivatives resemble the relaxed (R) or taut (T) state, respectively. In the present study, we attempt to characterize the conformation of these derivatives and the effects of ligands. The putative T-state derivative was similar to the native enzyme in its reactivity towards p-hydroxymercuribenzoate and in the increase of reactivity upon addition of succinate. However, unlike the native enzyme it was not activated by succinate at low substrate concentrations. On the other hand, the putative R-state derivative showed greatly enhanced reactivity which was not substantially increased by succinate. In the presence of urea, the native enzyme and the two cross-linked derivatives all resembled the R state. Thus at low substrate concentrations urea activated both the native enzyme and the t-state derivative. In contrast, the effect of urea on the R state derivative is mainly inhibitory. The above results show that the R state has been definitely stabilized whereas the T-state derivative retains some conformational flexibility. Our observations also indicate that the conformational change induced by succinate has two distinct components of which only one is allowed in the T-state derivative.
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001386:Azides / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology; D006902:Hydroxymercuribenzoates / Q000378:metabolism; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D013386:Succinates / Q000494:pharmacology; D019802:Succinic Acid; D014508:Urea / Q000494:pharmacology
D001386:Azides; D003432:Cross-Linking Reagents; D006902:Hydroxymercuribenzoates; D013386:Succinates; C030601:4-hydroxymercuribenzoate; C011122:tartryl diazide; D014508:Urea; D019802:Succinic Acid; D001221:Aspartate Carbamoyltransferase