Jy. Li et al., SITE-DIRECTED MUTAGENESIS OF RABBIT MUSCLE PHOSPHOFRUCTOKINASE CDNA -MUTATIONS AT GLUTAMINE-200 AFFECT THE ALLOSTERIC PROPERTIES OF THE ENZYME, The Journal of biological chemistry, 268(33), 1993, pp. 24599-24606
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.