SITE-DIRECTED MUTAGENESIS OF RABBIT MUSCLE PHOSPHOFRUCTOKINASE CDNA -MUTATIONS AT GLUTAMINE-200 AFFECT THE ALLOSTERIC PROPERTIES OF THE ENZYME

Full-length cDNA for rabbit muscle phosphofructokinase has been cloned and characterized (Li, J., Chen, Z., Lu, L., Byrnes, M., and Chang, S . H. (1990) Biochem. Biophys. Res. Commun. 170, 1056-1060). The 2.8-ki lobase cDNA was inserted in the plasmid vector pPL2 and transformed in to Escherichia coli cells deficient in endogenous phosphofructokinase activity (DF 1020). The recombinant phosphofructokinase so prepared is nearly identical in kinetic properties and size of subunits to the en zyme isolated from rabbit muscle. On the basis of the sequence homolog y between the muscle and the bacterial phosphofructokinases and the cr ystallographic structure of the latter, the glutamine at position 200 of the muscle enzyme is implicated in the allosteric transitions. This residue was replaced by alanine (Q200A), glutamate (Q200E), or argini ne (Q200R). The purified enzymes were analyzed for quaternary structur e, activity, and allosteric properties. The native and all the altered enzymes are tetramers. At pH 7.0, the wild-type enzyme is sensitive t o inhibition by ATP at concentration above 0.6 mM, and its activity re sponds to fructose 6-phosphate concentration cooperatively at high ATP concentration. In contrast, the mutated enzyme Q200R is virtually ins ensitive to ATP inhibition up to 7 mM. Thus at high ATP concentration, its activity responds to fructose 6-phosphate concentration in a mann er similar to the activated form of the native enzyme. Under the same conditions, mutant Q200E exhibits cooperative behavior only at much hi gher concentration of fructose 6-phosphate. Mutant Q200A is active at pH 8.0 but inactive at pH 7.0. The native enzyme and all three mutants are activated by inorganic phosphate and fructose 2,6-bisphosphate an d inhibited by citrate.