A calorimetric study of the interaction of pyridoxal 5'-phosphate with aspartate apoaminotransferase and model compounds.
Schiff base formation between pyridoxal 5'-phosphate and model compounds of increasing complexity (i.e. L-valine and poly-L-lysine) and between pyridoxal 5'-phosphate and the apoenzyme of aspartate aminotransferase has been analyzed by microcalorimetric methods. The apparent pKa values and protonation enthalpy values for the relevant groups ionizing in the pH 4-9 range have been determined for the Schiff bases of L-valine and of poly-L-lysine. Upon Schiff base formation, the only noticeable change is the lowering of the ring nitrogen pK, accompanied by an increase of the relative delta H. In the poly-L-lysine Schiff base, however, the delta H relative to the protonation of the phosphate dianion becomes more negative. This behavior suggests a multiple interaction between the polymer and the ligand. The intrinsic heat of formation is small (congruent to -1 kcal/mol), of the same order of magnitude for both Schiff bases, and appears to be independent of the nature of the aminic reagent. The heat of reaction of pyridoxal 5'-phosphate with aspartate apoaminotransferase has been determined in the pH 6.2-8.8 range at 19 degrees C and at 25 degrees C. Each isotherm is characterized by a lack of proton evolution, a result that is unexpected on the basis of the known pK values of the reagents, and by a sharp pH dependence of the enthalpy change. Moreover, comparison of the two isotherms allows: (a) detection of a protonation-dependent effect (pK 7.5 at 25 degrees C), (b) exclusion of a preferential binding of the coenzyme to the apoenzyme in a particular ionization state; and (c) suggestion of a tightening of the protein molecule upon holoenzyme formation.
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D002151:Calorimetry; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D011199:Potentiometry; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D012545:Schiff Bases; D013552:Swine
D001051:Apoenzymes; D012545:Schiff Bases; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases