Temperature- and time-dependent inactivation of pyrroline-5-carboxylate synthase: suggestive evidence for an allosteric regulation of the enzyme.
When pyrroline-5-carboxylate (PC) synthase activity in the membrane of mitochondria of rat small intestine mucosa was assayed in the presence of 0.5 mM ornithine, the time course of inactivation showed that the activity disappeared entirely by about 8 min at 30 degrees C, whereas there was no decrease in the activity at 15 degrees C. A prior incubation of the enzyme with ornithine at 30 or 37 degrees C in the presence of 50% sorbitol as a thermal stabilizer resulted in a marked loss of the activity, while that at 0 or 15 degrees C did not lose any. This suggests that PC synthase is inactivated by ornithine regardless of the presence of substrates. The inactivation at 30 degrees C proceeded gradually for about 7 h, until an equilibrium was attained. Extensive dialysis allowed the inactivated enzyme to regain about 60% of the original activity. These results suggest that the inactivation is reversible. The concentration of ornithine and the percentage of inactivation at equilibrium was correlated by the Hill equation and displayed a sigmoidicity with n = 1.47 and [S]50 = 0.036 mM. In the presence of sorbitol, the inactivation was prevented by 0.2 mM ATP or ADP. The role of the nucleotides in PC synthase regulation is discussed.
D000227:Adenine Nucleotides / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000201:enzymology; D007700:Kinetics; D008297:Male; D009952:Ornithine / Q000494:pharmacology; D009953:Ornithine-Oxo-Acid Transaminase / Q000037:antagonists & inhibitors*; D051381:Rats; D011919:Rats, Inbred Strains; D013012:Sorbitol / Q000494:pharmacology; D013367:Submitochondrial Particles / Q000201:enzymology; D013696:Temperature; D000637:Transaminases / Q000037:antagonists & inhibitors*
D000227:Adenine Nucleotides; D013012:Sorbitol; D009952:Ornithine; D000637:Transaminases; D009953:Ornithine-Oxo-Acid Transaminase