Document Detail


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.
MedLine Citation:
PMID:  2168419     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
A Tauler; K Lin; S J Pilkis
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  265     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1990 Sep 
Date Detail:
Created Date:  1990-10-11     Completed Date:  1990-10-11     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  15617-22     Citation Subset:  IM    
Affiliation:
Department of Physiology and Biophysics, State University of New York, School of Medicine, Stony Brook 11794-8661.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Base Sequence
Binding Sites
Escherichia coli / genetics
Histidine*
Kinetics
Liver / enzymology*
Models, Structural
Molecular Sequence Data
Multienzyme Complexes / genetics*,  metabolism
Mutation*
Oligonucleotide Probes
Phosphofructokinase-2
Phosphoric Monoester Hydrolases / genetics*,  metabolism
Phosphorylation
Phosphotransferases / genetics*,  metabolism
Recombinant Proteins / metabolism
Sequence Homology, Nucleic Acid
Grant Support
ID/Acronym/Agency:
DK 38354/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Multienzyme Complexes; 0/Oligonucleotide Probes; 0/Recombinant Proteins; 71-00-1/Histidine; EC 2.7.-/Phosphotransferases; EC 2.7.1.105/Phosphofructokinase-2; EC 3.1.3.-/Phosphoric Monoester Hydrolases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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