Conformational properties of pig-heart cytoplasmic aspartate aminotransferase. Circular-dichroism and absorption-spectroscopic study of dicarboxylate binding.
1. The interaction between aspartate aminotransferase and dicarboxylates of various chain lengths and geometries has been studied from pH 6.5 TO 8.5 by circular dichroism (CD) and absorption spectroscopy. Liganding causes protonation of the pyridoxal phosphate-enzyme Schiff's base complex; the consequent changes in optical properties deltaAlambda, deltaCDlambda at the coenzyme maxima (lambda = 363 or 430 nm) are analysed for binding constants and the degree of perturbation of the coenzyme protonic dissociation constant, pKa. 2. Aliphate dicarboxylates follow linear binding functions for all optical parameters; in contrast, m and p-phthalates follow non-linear binding functions for both deltaAlambda and deltaCDlambda, implying that successive phthalate ligands bind with decreasing affinity. The ratio detlaCDlambda is effectively constant for a given ligand and the characteristic values for aromatic ligands indicate a changed environment for the coenzyme. 3. Inspection of the non-linear process for phthalates suggests that initially, binding occurs with high affinity, but with characteristically small effects on pKa. It is inferred that alipathic and aromatic dicarboxylates bind at different subsites in the active site region, perturbing the coenzyme pKa by an indirect protein-mediated mechanism. 4. Non-linearity of binding could derive from multiple binding to an individual subunit. Alternatively, different single sites may exist on adjacent subunits of the dimer, implying non-equivalence between otherwise identical subunits, expressed in properties involving groups close to the active site.
D000818:Animals; D001219:Aspartate Aminotransferases; D001665:Binding Sites; D002942:Circular Dichroism; D003593:Cytoplasm / Q000201:enzymology; D003998:Dicarboxylic Acids; D007700:Kinetics; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013552:Swine
D003998:Dicarboxylic Acids; D001219:Aspartate Aminotransferases