Ligand binding-promoted conformational changes in yeast ornithine transcarbamoylase.
It has been proposed that regulatory multienzyme complex formation between yeast ornithine transcarbamoylase (OTCase) and arginase is triggered by a conformational change promoted by the binding of ornithine to a regulatory site in OTCase (Wiame, J.-M. (1971) Curr. Top. Cell. Regul. 4, 1-38). To isolate the binding of ornithine to the proposed regulatory site, the active site was blocked with the high affinity (Ki = 13 +/- 1.4 nM) bisubstrate analogue, delta-N-phosphonacetyl-L-ornithine (PALO). The binding of PALO to the active site produces large changes in the absorption (delta A290-296 = 0.010/mg of enzyme) and in the fluorescence (25% quenching) of the protein. These changes both saturate at one PALO/polypeptide chain. The binding of PALO also changes the rate constant for diffusional acrylamide quenching by 43% and increases the midpoint for the thermal denaturation of the enzyme by 13 degrees C. Finally, PALO binding results in a +2.8% change in the sedimentation coefficient demonstrating that these spectral and energetic changes are associated with a gross structural change in the enzyme. In an effort to detect ligand binding to the proposed effector site on OTCase, ornithine was added to the enzyme saturated with PALO, and consequent conformational changes were tested for using methodologies identical to those which demonstrated active site ligand binding-promoted conformational changes. In no instance were any additional differences observed. Hence, strong support for isosteric effector binding-promoted conformational changes cannot be presented. We conclude that active site ligand binding events themselves are responsible for conformational changes which promote enzyme-enzyme association of OTCase with arginase.
D001665:Binding Sites; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D008024:Ligands / Q000378:metabolism; D008433:Mathematics; D009952:Ornithine / Q000031:analogs & derivatives / Q000378:metabolism; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013053:Spectrophotometry
D006146:Guanidines; D008024:Ligands; C020464:N(delta)-(phosphonoacetyl)-L-ornithine; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase; D019791:Guanidine