The kinetic properties of adenylate deaminase from human erythrocytes

Abstract

Adenylate deaminase (AMP aminohydrolase, EC 3.5.4.6) was purified 500-fold from the human erythrocytes by DEAE-cellulose adsorption and chromatography on hydroxylapatite. Its isoelectric point is 5.0 and the pH of optimal activity is near 7.0. Although the enzyme is markedly activated by ATP and by monovalent cations, it is able to deaminate AMP in the absence of activators. At pH 7 the order of effectiveness for activation by monovalent cations is K + > Rb + > Li + > Na + > Cs +. The presence of ATP or changes in pH somewhat alter their relative activities but not the order. dATP activates the enzyme as effectively as ATP. ADP activates to a lesser degree and GTP is without effect. The enzyme displays a cooperative effect with substrate and also with its activators, monovalent cations and ATP. The activators thus increase the apparent affinity of the enzyme for AMP without affecting the maximum reaction velocity. When both cation and ATP are added, the substrate-velocity curve is changed from a pronounced sigmoidal curve to a rectangular hyperbola. The apparent affinity for AMP increases as the concentration of cation is raised. Conversely, the affinity for cation increases when the concentration of AMP is elevated or ATP is added. Halide anions are non-competitive inhibitors and decrease in their effectiveness as follows: F − > I − > Br > Cl − . P i and 2,3-diphosphglyceric acid inhibit the enzyme competitively and ATP counteracts that inhibition. In the presence of 1 mM ATP and 100 mM KCl the K i values for P i and 2,3-diphosphoglyceric acid are between 4 and 11 mM.