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 Ser7 (mutant enzyme RT2KS7, where RT2K = rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), Ser15 (RT2KS15), or Ser30 (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 Km values of protein kinase A for RT2KS7, RT2KS15, and RT2KS30 were 250 microM, 110 microM, and 50 microM, 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 RT2KS15 and RT2KS7 showed an 8-fold increase in KmFru6P, an 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 KmFru6P 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.(ABSTRACT TRUNCATED AT 250 WORDS)