Kinetics of Phosphate Incorporation into Adenosine Triphosphate and Guanosine Triphosphate in Bacteria

Escherichia coli cells were labeled with 33PO4 for several generations and then 32PO4 was added, samples were taken at various times, and adenosine triphosphate (ATP) and guanosine triphosphate (GTP) were purified from an acid extract. The ratio of 32P/33P in each of the six phosphate species was determined and compared to the ratio expected at isotopic steady state. The rate constants of the simplest network of pools which could explain the data, and were consistent with metabolic pathways, were then calculated. In the range of times studied (30 to 3,000 s at 21.5 C) the following generalizations can be made. (i) The γ-phosphate at ATP requires 2,200 s to achieve 50% of its final specific activity and behaves as if it were exchanging with another species 4.4 times as large, with this other species turning over in 540 s. (ii) The β-phosphate of ATP and the γ-phosphate of GTP are in equilibrium with the γ-phosphate of ATP. (iii) The specific activity of the γ-phosphate of GTP lags 66 s behind the γ-phosphate of ATP. (iv) The α-phosphates of ATP and GTP have equal specific activities which lag far behind the other four phosphates. A single precursor pool with a turnover time of 416s fits the data best, although a fair fit can be obtained assuming several sequential precursor pools with much shorter turnover times. These findings are consistent with known biosynthetic pathways and the probable flows through them, including the relationship of nucleotide biosynthesis to ribonucleic acid synthesis and turnover.