Identification of active site residues in pyrophosphate-dependent phosphofructo-1-kinase by site-directed mutagenesis.
The primary structure of pyrophosphate-dependent phosphofructokinase (PFK) from Propionibacterium freudenreichii exhibits a low but significant level of sequence identity with Escherichia coli ATP-dependent PFK, permitting the tentative assignment of residues that may be involved in catalysis. Based on these assignments, the roles in catalysis of 2 aspartyl residues (Asp151 and Asp153) and 2 lysyl residues (Lys80 and Lys85) were examined. Mutagenesis of the Asp151 to alanine and serine reduced kcat by a factors of 2 x 10(4) and 4 x 10(5), respectively, while showing no change in Km for either substrate in the forward reaction or for metal ion in the back reaction. The kcat for Asp153 was decreased by a factor of 700 with no change in Km for pyrophosphate and an increase of about 20-fold in Km for fructose 6-P and close to 4-fold for magnesium ion. That these changes in the mutants were not the result of global conformational changes was indicated by their identical behavior during substrate-specific elution chromatography, ion-exchange chromatography, and limited proteolysis by trypsin and subtilisin. Mutations of Lys80 and Lys85 showed no significant changes in kinetic parameters, suggesting no involvement in mechanism or substrate binding. These and other results permit preliminary modeling of the active site of pyrophosphate-dependent PFK.
Green
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Department of Biological Chemistry, University of Health Sciences, Chicago Medical School, Illinois 60064.
Tripathi
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Kemp
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D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D008670:Metals; D001224:Aspartic Acid; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; D008239:Lysine