Abstract
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-kilobase cDNA was inserted in the plasmid vector pPL2 and transformed into 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 enzyme isolated from rabbit muscle. On the basis of the sequence homology between the muscle and the bacterial phosphofructokinases and the crystallographic 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 arginine (Q200R). The purified enzymes were analyzed for quaternary structure, activity, and allosteric properties. The native and all the altered enzymes are tetramers. At pH 7.0, the wild-type enzyme is sensitive to inhibition by ATP at concentration above 0.6 mM, and its activity responds to fructose 6-phosphate concentration cooperatively at high ATP concentration. In contrast, the mutated enzyme Q200R is virtually insensitive to ATP inhibition up to 7 mM. Thus at high ATP concentration, its activity responds to fructose 6-phosphate concentration is a manner similar to the activated form of the native enzyme. Under the same conditions, mutant Q200E exhibits cooperative behavior only at much higher 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 and inhibited by citrate.
Highlights
The 2.8-kilobase cDNA was inserted in the plasmid its enzymology and its protein chemistr(yreviewed by Uyeda, vector pPL2 and transformed into
The recombinant phosphofructokinase so prepared is nearly identical in kinetic properties and size of subunits to the enzyme isolated from rabbit muscle
PFK cDNAs were completely sequenced, and itwas confirmed that no unintentional mutations hbaeden introduced into the remaining sequence of each cDNA
Summary
PFK activity (V),the enzyme was assayed in 1ml of reaction mixture containing 50 mM Tris-HC1, pH 8.0, 5 mM MgCl,, 1 mM DTT, 0.2 mM NADH, 10 mM Fru-6-P, 1 mM ATP, 1 mM Fru-2,6-Pz,and the following auxiliary enzymes: aldolase, 40 pg/ml; glycerophosphate dehydrogenase, 24 pg/ml; and triose phosphate isomerase, 2 pg/ml. The 0.6% gel at pH 7.0 and 0.8% gel at pH 8.0were stained directly for PFK activity using the method described by Brock (1969).In brief, the gel was soaked in 30 ml of solution containing 50 mM Tris-HC1,pH 8.2, 10 mM NanAs04,1mM EDTA, 4 mM MgC12,1 mM Fru-6-P, 1mM ATP, 1mM NAD, 0.074 mg/ml phenazine methosulfate, 0.4 mg/ml nitro blue tetrazolium chloride, 0.12 mg/ml glyceraldehyde-3-phosphate dehydrogenase, 0.1 mg/ml aldolase, and 7 pg/ml triose phosphate isomerase for 1.5 h at 37 "C followedby destaining in 10% acetic acid. Gel run at pH 7.0was soaked in 20 mM Tris phosphate, pH 8.0, prior to activity staining
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