Mechanism of modulation of rat liver fructose-2,6-bisphosphatase by nucleoside triphosphates
Abstract
The mechanism of modulation of fructose-2,6-bisphosphatase of rat liver 6- phosphofructo-2-kinase/fructose-2,6-bisphosphatase by nucleoside triphosphates was studied by employing the Escherichia coli-expressed bisphosphatase domain and a COOH-terminal 30-amino acid truncated form. These forms had K(m) values for substrate and K(i) values for products which were similar to those of the bisphosphatase of the intact bifunctional enzyme, but turnover numbers were 5-fold higher. All forms also exhibited substrate inhibition that was relieved by GTP and ATP. The nucleoside triphosphates bound to the active site, since they were competitive inhibitors at subsaturating substrate concentrations. Guanosine was also a competitive inhibitor at subsaturating substrate concentrations but did not activate at saturating substrate. ATP and GTP had K(d) values of 467 and 110 μM, respectively, and 1 mol of nucleoside triphosphate/mol bound per mol of bisphosphatase. The K(i) values for guanosine of two mutants, Lys356 → Ala and Arg360 → Ala, were unchanged from that of the wild-type enzyme. However, the K(i) for GTP for Arg360 → Ala was 17-fold higher than that of the wild-type enzyme, whereas that for Lys356 → Ala was unchanged. It was concluded that 1) nucleoside triphosphate modulation of the bisphosphatase of the bifunctional enzyme involves a direct interaction with the active site of the bisphosphatase domain; and 2) the activation is caused by the phosphate moieties of GTP and ATP competing with the 2-phospho group of fructose-2,6-bisphosphate for the phosphoenzyme intermediate, thus relieving substrate inhibition.