Succinyl Coenzyme A Synthetase of Escherichia coli: EFFECTS OF PHOSPHOENZYME FORMATION AND OF SUBSTRATE BINDING ON THE REACTIVITY AND STABILITY OF THE ENZYME

Abstract

Abstract Formation of phosphohistidine at only one of an apparent total of two active sites in Escherichia coli succinyl coenzyme A synthetase results in profound changes in the reactivity of the enzyme. On storage, the dephosphorylated form of enzyme loses activity at a rate approximately 100 times that of the monophosphorylated form. Similarly, there is a difference of two orders of magnitude in the rates of inactivation by trypsin of the phosphorylated and dephosphorylated enzymes. There is direct correlation between both the rate and extent of inactivation and the status of phosphorylation of the enzyme. In keeping with the ability of substrates to interconvert the phosphorylated and dephosphorylated forms of enzyme, ATP protects the previously dephosphorylated enzyme from proteolytic attack, whereas the presence of succinate and Mg++ renders previously phosphorylated enzyme susceptible to proteolysis. These effects are observed not only with trypsin, but with a variety of proteases having different specificities. The results indicate that phosphorylation of 1 active site histidine residue per oligomeric enzyme molecule results in a significant change in the three-dimensional structure of the enzyme to a tighter conformation, with concomitant stability and, perhaps, distortion of the second active site in the molecule so that its phosphorylation is unfavorable.