Site-directed mutagenesis of the fructose 6-phosphate binding site of the pyrophosphate-dependent phosphofructokinase of Entamoeba histolytica.
Attempts to define the active site of pyrophosphate-dependent phosphofructokinase (PPi-PFK) using homology modeling based on the three-dimensional structure of the ATP-dependent PFKs from bacteria have been frustrated by low sequence identity between PPi- and ATP-PFKs in their carboxyl terminal halves. In the current study, alanine scanning mutagenesis of residues in the carboxyl terminal half of the PPi-PFK of Entamoeba histolytica coupled with comparative sequence analysis and computational modeling is used to identify residues that contribute to fructose 6-phosphate (fructose 6-P) binding. Of seven alanine mutants that were generated by site-directed mutagenesis, Arg377, Ser392, Arg405, Lys408, His415, His416, and Arg423, only the last mutant, Arg423Ala, was found to have dramatically lower affinity for fructose 6-P. Mutation of Arg 423 decreased k(cat) by 10,000-fold and decreased apparent affinity for fructose 6-P by 126-fold, while the K(m) for PPi increased only 4-fold. The second greatest effect was seen with Arg377Ala, which had a nearly 10-fold decrease in apparent affinity and an approximate 60-fold decrease in maximal activity. Another residue, Tyr420, was chosen for mutagenesis by its complete identity in all other PPi-PFK. This residue and its homologue in Escherichia coli ATP-PFK, His249, were mutated and shown to be very important for substrate binding in both enzymes.
Deng
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Z
Department of Microbiology and Immunology, The Chicago Medical School, North Chicago, Illinois 60064, USA.
Wang
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X
Kemp
R G
RG
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004748:Entamoeba histolytica / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D005636:Fructosephosphates / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D005636:Fructosephosphates; C027618:fructose-6-phosphate; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; D000409:Alanine