ADENOSINE 5'-TETRAPHOSPHATE IS SYNTHESIZED BY THE HISTIDINE ALPHA-142-]ASPARAGINE MUTANT OF ESCHERICHIA-COLI SUCCINYL-COA SYNTHETASE

Recently, we described the properties of a mutant (H142N) of Escherichia coli succinyl coenzyme A (CoA) synthetase in which His-142 of the alpha-subunit was changed to Asn (Luo, G.-X., and Nishimura, J. S. (1991) J. Biol. Chem. 266, 20781-20785). The mutant enzyme was practically devoid of ability to catalyze the overall reaction but was able to catalyze half-reactions at significant rates. Thus, phosphorylation by ATP and dephosphorylation by ADP of the mutant enzyme occurred at rates that were at least 10 times greater than those with wild type enzyme, and dephosphorylation by succinate plus CoA (succinyl-CoA formation) proceeded with a V(max) of 10% that of wild type, with no change in K(m) for succinate and very little change in K(m) for CoA. In the present work, it has been shown that incubation of P-32-labeled H142N with ATP caused a rapid depletion of label from the enzyme and incorporation of radioactivity into a nucleotide species that was neither ATP nor ADP. This reaction was catalyzed at comparatively negligible rates by wild type enzyme. Analysis of the labeled product by high pressure liquid chromatography and P-31 NMR revealed that it was adenosine 5'-tetraphosphate (AP4). Incubation of labeled H142N with the ATP analog beta, gamma-methylene adenosine triphosphate also gave a product that appeared to be the corresponding tetraphosphate. The reaction in which AP4 was formed was greatly stimulated by the addition of phosphoenolpyruvate plus pyruvate kinase and strongly inhibited by ADP and by CoA plus succinate. The results are consistent with binding of ATP to, and reaction with, phosphorylated succinyl-CoA synthetase to form AP4. In this reaction, it was determined that the K(m) for ATP and the turnover number of phosphorylated enzyme were 14.5-mu-M and 0.024 s-1, respectively.