Effects of cobaltous ion on various catalytic parameters and on heterologous subunit interactions of Escherichia coli glutamine synthetase

Abstract

Cobaltous ion, like Mg 2+, has been found to support biosynthetic activity of the unadenylylated form [E (0)] of glutamine synthetase from Escherichia coli, but not the adenylylated form [E (12)]. The characteristics of Co 2+-activated enzyme however, are significantly different from those of the enzyme activated by Mg 2+. The Co 2+ enzyme has a symmetrical pH-activity profile with an optimum at pH 7.5, whereas the profile of Mg 2+ enzyme is asymmetrical with an optimum at pH 9.0. The activity of Mg 2+ enzyme is a normal hyperbolic function of either glutamate or NH 4 + concentration, but the activity of Co 2+ enzyme is a complex function of either substrate concentration. Activity of the enzyme increases sharply in a sigmoidal manner with increasing Co 2+ concentration to a maximal value and then declines sharply with a further increase in Co 2+ concentration. The magnitude of maximal activity and the concentration of Co 2+ required to obtain maximal activity are both dependent upon the concentrations of ATP and of glutamate. Whereas Mg 2+-supported activity is inhibited by alanine at all levels of substrate examined, the Co 2+-supported activity is stimulated by low concentrations of alanine and is inhibited by high concentrations of this effector when the levels of glutamate and NH 4 + are not saturating. When saturated with both substrates, alanine is an inhibitor at all concentrations. In contrast to the sigmoidal relationship between state of adenylylation and the specific activity of Mg 2+ enzyme (8) the specific activity in the presence of Co 2+ is a relatively simple nonlinear function of the average state of adenylylation. The results indicate that in the presence of Co 2+, heterologous interactions between adenylylated and unadenylylated subunits within hybrid enzyme molecules (i.e., molecules containing both adenylylated and unadenylylated subunits) affect the catalytic potential of unadenylylated subunits. The data are consistent with the view that activity in the presence of Co 2+ is restricted to unadenylylated subunits that are not in direct contact with adenylylated subunits in the same hexagonal ring. That heterologous subunit interactions affect catalytic potential is also illustrated by the fact that at low substrate concentrations the percentage activation by alanine is a nonlinear function of the average state of adenylylation; it is a linear function of the calculated number of unadenylylated subunits not in contact with adenylylated subunits.