Inactivation of wheat-germ aspartate transcarbamoylase by the triazinyl dye, procion red HE3B.
Aspartate transcarbamoylase from wheat germ is irreversibly inactivated by the triazinyl dye Procion Red HE3B. Since triazinyl dyes may mimic nucleotides, and UMP is a known allosteric modifier of this enzyme, the reaction was studied to elucidate whether the dye is an 'affinity label' for the enzyme. The reaction is apparently first order in the first 5-10 min, but is more complex in the longer term and does not go to completion. Kinetic analysis of the initial phase suggests that there are two parallel reactions, one saturable (dye binds reversibly before reaction) and one non-saturable (biomolecular). The apparent rate constant kapp (i.e. the sum of the rate constants for the parallel reactions) varies only slightly over the pH range 7-10. In the presence of a number of active centre ligands, as well as the allosteric ligand UMP, there is a clear increase in kapp. This finding is contrary to the reduction in rate of inactivation (protection) normally provided by ligands against active-site directed reagents, suggesting that in the saturable reaction, there is a conformational change upon dye-binding that increases the exposure of the essential residue(s) with which the dye reacts. These results show that, although it probably inactivates by reaction with specific amino-acid residues, the dye is not bound at the substrate-binding or allosteric sites, i.e. it is not an affinity-labeling reagent in the usual sense.
Easton
M J
MJ
School of Biological and Chemical Sciences, Thames Polytechnic, London, U.K.
Yon
R J
RJ
D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D012639:Seeds / Q000201:enzymology*; D014227:Triazines / Q000378:metabolism*; D014908:Triticum / Q000201:enzymology*; D014542:Uridine Monophosphate / Q000378:metabolism
D014227:Triazines; C011535:procion brilliant red H 3B; D014542:Uridine Monophosphate; D001221:Aspartate Carbamoyltransferase