Fluorescent probe study with 1-anilino-8-naphthalene sulfonate on acetylglutamate activation, ATP binding and conformational changes of the rat liver carbamoyl-phosphate synthetase.
The activity and stability of carbamoyl-phosphate synthetase (EC 6.3.4.16) may involve hydrophobic and ionic bonds within the enzyme. The 1-anilino-8-naphthalene sulfonate (ANS) equilibrium binding method with hydrophobic and ionic sites in enzymes, therefore, seemed suitable for the study of the acetylglutamate activation and ATP binding of the enzyme. The enzyme had a high affinity for the dye but low fluorescent yields. The enzyme had 32-88 ANS binding sites, depending on combination with ATP and acetylglutamate, and individual affinity constants for each combination. Despite the large number of binding sites, the acetylglutamate and ATP concentrations for half-maximal fluorescent change (10-40 microM) corresponded to the high-affinity bound ATP (ATPB) and acetylglutamate Kd values. In kinetic studies, ANS competed with ATP or acetylglutamate. The extrapolated ANS Ki values for ATP or acetylglutamate were both 35 microM. This value agreed with the ANS Kd value of the enzyme X ATP conformation, indicating that this was the conformation competed for by ANS. Since ANS did not influence the HCO3-dependent ATPase, ANS was concluded to compete with the ATPB binding conformation and transitional changes. This study suggests that part of the activator role of acetylglutamate may be to change the tertiary structure of the enzyme to induce hydrophobic sites which are accessible to ANS and possibly at the ATPB site.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D000818:Animals; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D004789:Enzyme Activation; D005971:Glutamates / Q000494:pharmacology*; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D005971:Glutamates; C027132:1-anilino-8-naphthalenesulfonate; D000255:Adenosine Triphosphate; D008025:Ligases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); C016195:N-acetylglutamic acid