Interaction of ligands with pig heart citrate synthase: conformational changes and catalysis.
The fluorescence polarization of 8-hydroxypyrene (1,3,6)trisulfonate (HPT) increases upon interaction with pig heart citrate synthase. Titration of HPT with increasing concentrations of citrate synthase exhibits a hyperbolic saturation behavior, from which the dissociation constant of the enzyme-HPT complex (3.64 +/- 0.3 microM) was determined. The enzyme-HPT interaction is competitively inhibited by oxaloacetate (but not affected by acetyl CoA) with a Ki of 4.3 +/- 1.8 microM. This value is similar to the dissociation constant (Kd = 4.5 +/- 1.6 microM) for the enzyme-oxalocetate complex (determined in the absence of any effector ligand), as well as to the Km for oxaloacetate (3.9 +/- 0.7 microM) in a steady-state citrate synthase catalyzed reaction at a saturating concentration of acetyl CoA. However, the dissociation constant for the citrate synthase-oxaloacetate complex determined by the urea denaturation method is at least 25-fold lower than those determined by the other methods. This suggests an effector role of urea in strengthening the enzyme-oxaloacetate interaction. At low nondenaturing concentrations, urea inhibits the citrate synthase catalyzed reaction in an uncompetitive manner with respect to oxaloacetate, i.e., the Km for oxaloacetate decreases with an increase in urea concentration. This further suggests that urea stabilizes the interaction between citrate synthase and oxaloacetate. The effect of urea is specific for the substrate oxaloacetate, and not for the substrate analogue, HPT, although both these ligands bind citrate synthase with equal affinities, and protect the enzyme against thermal denaturation with equal magnitudes. The results presented herein are discussed in the light of known conformational states of the enzyme.
Johnson
J K
JK
Department of Biochemistry, North Dakota State University, Fargo 58105.
Srivastava
D K
DK
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000818:Animals; D001190:Arylsulfonates / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002950:Citrate (si)-Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D009206:Myocardium / Q000201:enzymology*; D010071:Oxaloacetates / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D013552:Swine; D014508:Urea / Q000494:pharmacology
D001190:Arylsulfonates; D005456:Fluorescent Dyes; D010071:Oxaloacetates; D000105:Acetyl Coenzyme A; D014508:Urea; D002950:Citrate (si)-Synthase; C005047:pyranine