Binding of substrates to aspartate aminotransferase. Evidence for a dissymmetrical binding

The binding of substrates L-glutamate and 2-oxoglutarate to aspartate aminotransferase was studied by spectrophotometric titration according to Jenkins and D'Ari [J. Biol. Chem. 241 (1966) 2845-2854]. Our data, obtained over a wide range of substrate concentrations, were not consistent with the hypothesis of independent and equivalent binding sites. Two other possibilities were considered: (a) the sites are independent but not equivalent; (b) a negative cooperativity occurs between the sites. It is possible to distinguish between these two hypotheses because the substrate binding is complex, with a couple of substrates and two forms of enzyme in equilibrium with the enzyme-substrate complexes. Distinct equations delta A/Et=f[substrates], where delta A = change in absorbance upon addition of substrates to the enzyme and Et = total site concentration, were derived for each case. The data were directly fitted to these equations by an iterative multilinear regression analysis, and the equilibrium constants were computed. This analysis showed that the binding sites are independent and not equivalent, with dissociation constants for 2-oxoglutarate of 2 micro M and 280 micro M and dissociation constants for glutamate of 1.7 mM and 22 mM. The molar absorption coefficient of the binary complexes is 2250 M-1 cm-1.