EFFECT OF ADDING PHOSPHORYLATION SITES FOR CAMP-DEPENDENT PROTEIN-KINASE TO RAT TESTIS 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE

In contrast to liver and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, the testis isozyme lacks a phosphorylation site for cAMP-dependent protein kinase. In order to determine the effect of phosphorylation site location for the protein kinase on rat testis bifunctional enzyme, consensus amino acid sequences (RRXS) were added at different distances from the N-terminus by site-directed mutagenesis. The expressed wild-type enzyme (WT) and mutant enzymes containing a phosphorylation site at Ser(7) (mutant enzyme RT2KS7, where RT2K = rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), Ser(15) (RT2KS15), or Ser(30) (RT2KS30) were purified to apparent homogeneity. All the mutant enzymes served as substrates for the protein kinase, and the phosphate incorporation was over 90%. The K-m values of protein kinase A for RT2KS7, RT2KS15, and RT2KS30 were 250 mu M, 110 mu M, and 50 mu M, respectively, and the relative rates were 1, 8, and 23. Various kinetic parameters of dephospho and phospho forms of these enzymes were determined. The kinetic constants of the dephospho form of RT2KS30 were similar to those of WT, but those of RT2KS 15 and RT2KS7 showed an 8-fold increase in K-m(Fru6P), approximately 30% decrease in the Fru-6-P,2-kinase activity, and a 3-fold increase in fructose-2,6-bisphosphatase activity. Phosphorylation of RT2KS30 resulted in a shift in the Fru-6-P saturation curve from Michaelis-Menten kinetics to sigmoidal, with increased K-m(Fru6P) and activation of fructose-2,6-bisphosphatase. The kinetic constants of RT2KS15 and RT2KS7 were not altered by phosphorylation. All the mutant enzymes were more sensitive to heat inactivation than was WT. Furthermore, the phospho-RT2KS30 was more thermally labile than its dephospho form. A Stern-Volmer plot of iodide quenching of RT2KS30 tryptophan fluorescence was nonlinear, but those of the other mutant enzymes were linear. These results suggest that all tryptophans in the RT2KS15 and RT2KPS7 mutant enzymes are more exposed and accessible to iodide than RT2KS30.