Effect of aggregation on the kinetic properties of aspartate aminotransferase.

In this investigation the steady-state kinetic parameters of the alpha subform of aspartate aminotransferase (EC 2.6.1.1) were determined in 0.2 M Tris - HCl, pH 8.0, at 25 degrees C. The kinetic parameters for both the forward and reverse reactions were determined under conditions where the enzyme is monomeric, while only the steady-state parameters associated with the forward reaction could be determined under conditions where the enzyme is dimeric enzyme decreased relative to that of monomeric enzyme, 245 versus 360 s(-1) while the Km for aspartate increased, 3.3 versus 2.6 mM. No significant change in the Michaelis constant for ketoglutarate was observed. The steady-state parameters of dimeric enzyme are slightly altered in 0.1 M Na4 P2O7, pH 8.0, the catalytic center activity and Michaelis constant for ketoglutarate being slightly larger. From the dependence of the initial velocity on enzyme concentration the dissociation constant for the monomer-dimer equilibrium is estimated to be 2 - 10(-8) M. A similar value of the dissociation constant was estimated from Sephadex gel filtration experiments.