SITE-DIRECTED MUTAGENESIS OF RAT MUSCLE 6-PHOSPHOFRUCTO-2-KINASE FRUCTOSE-2,6-BISPHOSPHATASE - ROLE OF ASP-130 IN THE 2-KINASE DOMAIN/

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (P FK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to stu dy its role in catalysis and/or substrate binding. The D130A mutant di splayed a 30- to 140-fold decreased 2-kinase V-max., depending on the pH, and a 30- and 60-fold increase in K-m for MgATP and Fru-6-P respec tively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 t o Ala had no effect on the 2-phosphatase activity, and fluorescence me asurements indicated that the changes in kinetic properties of PFK-2 i n the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.