Abstract
The effect of metal ions on the catalytic and thermodynamic properties of the aminopeptidase isolated from pronase has been investigated. A decrease in K and enhanced activity were observed for most of the metal ions examined. Ca(II) exhibits the most prominent effect on enzyme activity. The observed stability constants for the enzyme-bound metal ions are in the range of 10 -10 M , which is much smaller than that of a metalloenzyme, indicating that the metal ion is not an integral part of the enzyme. The complexation of l-leucine-p-nitroanilide and the transition metal ions causes a reduction in free substrate concentration and hence a concomitant decrease in enzyme activity. Therefore, care must be taken to account for this decrease in substrate concentration in order to obtain reliable kinetic parameters. Binding of E and S to form ES was accompanied by a decrease in Gibbs free energy, whereas a dramatic increase in the free energy was observed for the conversion of ES to ES . Both the enthalpy and the entropy were found to be crucial in destabilizing ES . In the presence of Ca(II), ES is stabilized by ∼ 1 kcal/mol and ES by ∼ 1.4 kcal/mol. The stabilization of ES by the presence of Ca(II) is reflected by a smaller K value compared to that of the metal-free enzyme. The activation free energies for the process E + S → ES were 10.8 and 9.4 kcal/mol for the metal-free and the Ca(II)-activated enzymes, respectively. The difference of ∼ 1.4 kcal/mol in the activation free energy may account for enhanced activity by the presence of Ca(II). © 1995.