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.