MECHANISM OF MODULATION OF RAT-LIVER FRUCTOSE-2,6-BISPHOSPHATASE BY NUCLEOSIDE TRIPHOSPHATES

The mechanism of modulation of fructose-2,6-bisphosphatase of rat live r phosphofructo-2-kinase/fructose-2,6-bisphosphatase by nucleoside tri phosphates was studied by employing the Escherichia coli-expressed bis phosphatase domain and a COOH-terminal 30-amino acid truncated form. T hese forms had K(m) values for substrate and K(i) values for products which were similar to those of the bisphosphatase of the intact bifunc tional 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 com petitive inhibitors at subsaturating substrate concentrations. Guanosi ne was also a competitive inhibitor at subsaturating substrate concent rations but did not activate at saturating substrate. ATP and GTP had K(d) values of 467 and 110 muM, respectively, and 1 mol of nucleoside triphosphate/mol bound per mol of bisphosphatase. The K(i) values for guanosine of two mutants, Lys36 --> Ala and Arg360 --> Ala, were uncha nged 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, w hereas that for Lys356 --> Ala was unchanged. It was concluded that 1) nucleoside triphosphate modulation of the bisphosphatase of the bifun ctional enzyme involves a direct interaction with the active site of t he bisphosphatase domain; and 2) the activation is caused by the phosp hate moieties of GTP and ATP competing with the 2-phospho group of fru ctose-2,6-bisphosphate for the phosphoenzyme intermediate, thus reliev ing substrate inhibition.