Identification of residues of Escherichia coli phosphofructokinase that contribute to nucleotide binding and specificity.
The apparent affinity of phosphofructo-1-kinase (PFK) of Escherichia coli for ATP is at least 10 times higher than for other nucleotides. Mutagenesis was directed toward five residues that may interact with ATP: Y41, F76, R77, R82, and R111. Alanine at position 41 or 76 increased the apparent Km by 49- and 62-fold, respectively. Position 41 requires the presence of a large hydrophobic residue and is not restricted to aromatic rings. Tryptophan and, to a lesser extent, phenylalanine could substitute at position 76. None of the mutants at 41 or 76 showed a change in the preference for alternative purines, although F76W used CTP 3 times better than the wild type enzyme. Mutations of R77 suggested that the interaction was hydrophobic with no influence on nucleotide preference. Mutation of R82 to alanine or glutamic acid increased the apparent Km for ATP by more than 20-fold and lowered the kcat/Km with ATP more than 30-fold. However, these mutants had a higher kcat/Km than wild type for both GTP and CTP, reflecting a loss of substrate preference. A loss in preference is seen as well with R111A where the kcat/Km for ATP decreases by only 68%, but the kcat/Km with GTP increases more than 10-fold. Activities with ITP, CTP, and UTP are also higher than with the wild type enzyme. Arginine residues at positions 82 and 111 are important dictators of nucleoside triphosphate preference.
Wang
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Department of Microbiology, The Chicago Medical School, Illinois 60064, USA.
Kemp
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D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003570:Cytidine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007293:Inosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D014443:Tyrosine / Q000235:genetics; D014544:Uridine Triphosphate / Q000378:metabolism
D000227:Adenine Nucleotides; D000596:Amino Acids; D007293:Inosine Triphosphate; D014443:Tyrosine; D010649:Phenylalanine; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D010732:Phosphofructokinase-1; D014544:Uridine Triphosphate