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 osphofructo-2-kinase/fructose-2,6-bisph osphatases, the testis isozyme lacks a phosphorylation site for cAMP-d ependent protein kinase. In order to determine the effect of phosphory lation 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 expre ssed wild-type enzyme (WT) and mutant enzymes containing a phosphoryla tion site at Ser(7) (mutant enzyme RT2KS7, where RT2K = rat testis osp hofructo-2-kinase/fructose-2,6-bisphosphatase), Ser(15) (RT2KS15), or Ser(30) (RT2KS30) were purified to apparent homogeneity. All the mutan t enzymes served as substrates for the protein kinase, and the phospha te 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, res pectively, and the relative rates were 1, 8, and 23. Various kinetic p arameters of dephospho and phospho forms of these enzymes were determi ned. The kinetic constants of the dephospho form of RT2KS30 were simil ar to those of WT, but those of RT2KS 15 and RT2KS7 showed an 8-fold i ncrease in K-m(Fru6P), approximately 30% decrease in the Fru-6-P,2-kin ase activity, and a 3-fold increase in fructose-2,6-bisphosphatase act ivity. Phosphorylation of RT2KS30 resulted in a shift in the Fru-6-P s aturation curve from Michaelis-Menten kinetics to sigmoidal, with incr eased K-m(Fru6P) and activation of fructose-2,6-bisphosphatase. The ki netic constants of RT2KS15 and RT2KS7 were not altered by phosphorylat ion. All the mutant enzymes were more sensitive to heat inactivation t han was WT. Furthermore, the phospho-RT2KS30 was more thermally labile than its dephospho form. A Stern-Volmer plot of iodide quenching of R T2KS30 tryptophan fluorescence was nonlinear, but those of the other m utant enzymes were linear. These results suggest that all tryptophans in the RT2KS15 and RT2KPS7 mutant enzymes are more exposed and accessi ble to iodide than RT2KS30.