title: Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii.
abstract: Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii was inactivated by low concentrations of the lysine-specific reagent pyridoxal phosphate (PLP) after sodium borohydride reduction. The substrates fructose 6-phosphate and fructose 1,6-bisphosphate protected against inactivation whereas inorganic pyrophosphate had little effect. An HPLC profile of a tryptic digest of PPi-PFK modified at low concentrations of PLP showed a single major peak with only a small number of minor peaks. The major peak peptide was isolated and sequenced to obtain IGAGXTMVQK, where X represents a modified lysine residue, corresponding to Lys-315. Lys-315 was protected from reaction with PLP by fructose 1,6-bisphosphate. As indicated by HPLC maps of PPi-PFK modified with varying concentrations of PLP, a direct correlation was observed between activity loss and the modification of Lys-315. Two of the minor peptide peaks were shown to contain Lys-80 and Lys-85, which were modified in a mutually exclusive manner. Partial protection against modification of these two residues was provided by MgPPi. The data were used to adjust the sequence alignment of the Propionibacterium enzyme with that of ATP-dependent PFK of Escherichia coli to identify homologous residues in the substrate binding site. It is suggested that Lys-315 interacts with the 6-phosphate of fructose 6-phosphate and that Lys-80 and -85 may be located near the pyrophosphate binding site.
authors.affiliation: Department of Biological Chemistry, University of Health Sciences, Chicago Medical School, Illinois 60064.
authors.forename: P C
authors.initials: PC
authors.lastname: Green
authors.forename: S P
authors.initials: SP
authors.lastname: Latshaw
authors.forename: U S
authors.initials: US
authors.lastname: Ladror
authors.forename: R G
authors.initials: RG
authors.lastname: Kemp
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D011756:Diphosphates / Q000737:chemistry*; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010732:Phosphofructokinase-1 / Q000737:chemistry; D010770:Phosphotransferases / Q000737:chemistry*; D011424:Propionibacterium / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000737:chemistry; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000187:drug effects; D014357:Trypsin
chemical_list: D011756:Diphosphates; D011732:Pyridoxal Phosphate; D010770:Phosphotransferases; D010732:Phosphofructokinase-1; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; D014357:Trypsin; D008239:Lysine