Expression of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. Role of N-2 proline in degradation of the protein.
Human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is 96% identical to the rat and bovine liver enzymes, and all of the critical catalytic and substrate binding residues in both the kinase and bisphosphatase domains are conserved in the three enzymes. However, in contrast to rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, which is readily expressed in an Escherichia coli T-7 RNA polymerase-based expression system, the human liver bifunctional enzyme could not be expressed in this system. Western blot and slot blot analysis revealed that although both the bifunctional enzyme protein and its mRNA were rapidly induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside, the protein underwent rapid degradation. Deletion of the N-2 proline residue or its mutation to arginine, the corresponding residue in the rat liver enzyme, revealed that this proline residue was responsible for its rapid degradation. The Pro-2-->Arg mutant could be expressed with a high yield (20 mg/liter) in E. coli. The results support the hypothesis that a proline residue at N-2 facilitates bifunctional enzyme degradation in E. coli. The E. coli expressed mutant form was purified to homogeneity by phosphocellulose chromatography, and its kinetic properties were compared with those of the rat liver enzyme. The kinetic properties of the two enzymes were identical except for the presence of substrate (fructose 6-phosphate) inhibition of the human liver enzyme but not of the rat liver enzyme. The ability to express and purify large amounts of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase will permit structure/function and x-ray crystal structure studies of the enzyme and ultimately its targeting for drug therapy.
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794-8661.
A J
AJ
Lange
L
L
Li
A M
AM
Vargas
S J
SJ
Pilkis
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011392:Proline; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D011392:Proline; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases