Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis.
The current model for hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase divides the protein into two functional domains: an N-terminal kinase domain and a carboxyl-terminal bisphosphatase domain. Site-directed mutagenesis was used to evaluate the role of two putative bisphosphatase active site histidyl residues in catalysis. His-258 has been implicated as a phosphoacceptor (Pilkis, S. J., Lively, M. O., and El-Maghrabi, M. R. (1987) J. Biol. Chem. 262, 12672-12675), and the importance of this residue was confirmed when it was mutated to alanine and neither bisphosphatase activity nor a phosphoenzyme intermediate could be detected. Mutation of His-392 to alanine produced an enzyme which had five percent of wild-type fructose 2,6-bisphosphatase activity, and the rate of phosphoenzyme formations was decreased from 4800 nmol/min/mg to 2.9 nmol/min/mg. Mutation of His-392 to phenylalanine, lysine, or aspartic acid also produced proteins that did not hydrolyze fructose 2,6-bisphosphate or form a phosphoenzyme intermediate. These results are consistent with an important role for His-392 in the bisphosphatase reaction, probably as a proton donor, and with its designation as an active site residue based on homology modeling (Bazan (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). H258A had the same Vmax for 6-phosphofructo-2-kinase as the wild-type enzyme, and the mutant's kinase was inhibited by cAMP-dependent phosphorylation. In addition, H392F and H392K did not catalyze the kinase reaction, although H392D had normal kinase activity which was also modulated by cAMP-dependent phosphorylation in the same manner as the wild-type enzyme. Thus, an active bisphosphatase domain is not a necessary condition for phosphorylation-induced changes in 6-phosphofructo-2-kinase activity. The results also suggest that structural and/or active site interactions exist between the two domains of the enzyme.
Tauler
A
A
Department of Physiology and Biophysics, State University of New York, School of Medicine, Stony Brook 11794-8661.
Lin
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K
Pilkis
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D015345:Oligonucleotide Probes; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D009097:Multienzyme Complexes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D006639:Histidine; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases