The involvement of proteolysis in conformational stability of the carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa.
The pyrimidine-3 gene of Neurospora crassa codes for a bifunctional enzyme catalysing the first two steps of the pyrimidine biosynthetic pathway. Difficulties have been experienced in purification due to the lability of the enzyme. The enzyme loses carbamoyl-phosphate synthetase (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) activity and undergoes a change in apparent molecular weight from the native 650,000 to 100,000 of the only detectable fragment. Attempts have been made therefore to stabilize the enzyme so as to minimise these effects. Elastinal, a protease inhibitor, reduces the effects, as do certain ultraviolet-sensitive mutant strains which lack a minor protease. The nature of the loss of carbamoyl-phosphate synthetase suggests an instability in the tertiary structure of the enzyme which can be reduced by the use of glycerol. Glycerol also exhibits a protease-inhibiting effect in this system. Although a range of protease inhibtors and use of uvs mutants can reduce the rate of decay of carbamoyl-phosphate synthetase activity, only glycerol can stabilize the native molecular weight. Our results support the hypothesis that the loss of carbamoyl-phosphate synthetase activity and change in molecular weight of the enzyme is a three-step sequence of proteolysis, conformational shift and cleavage of a further non-covalent bond.
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D004355:Drug Stability; D005990:Glycerol / Q000494:pharmacology; D008025:Ligases / Q000378:metabolism*; D009154:Mutation; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects
D009842:Oligopeptides; D011480:Protease Inhibitors; D001221:Aspartate Carbamoyltransferase; D010447:Peptide Hydrolases; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C010718:elastatinal; D005990:Glycerol