Identification of basic residues involved in substrate binding and catalysis by pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.
Despite overall low identity of sequence between ATP- and PPi-dependent phosphofructo-1-kinases (PFK), an alignment permits the tentative identification of catalytically important residues in PPi-dependent PFK. Seven basic residues of the PPi-dependent PFK of Propionibacterium freudenreichii, Arg-310, Lys-315, Arg-326, Arg-186, Arg-78, Arg-90, and Lys-148 were chosen on the basis of this alignment for site-directed mutagenesis to neutral residues. Mutants R186A, K315A, and R326A had substantial increases of 43-, 389- and 678-fold, respectively, in Km for fructose 6-phosphate relative to wild-type enzyme and relatively small effects on kcat. The modest kinetic effects of mutations at Arg-78, Arg-90, and Arg-310 were not considered to indicate important roles for these residues. On the other hand, mutant K148M had a Km for PPi that was increased by 132-fold and kcat lowered by a factor of 490. Cho and Cook (Cho, Y.-K., and Cook, P.F. (1988) Biochemistry 28, 4155-4160) concluded on the basis of kinetic and chemical modification data that the enzyme utilizes a proton shuttle mechanism and that two lysines are involved in substrate binding. The present data along with our previous data on mutant D151A support a double proton shuttle mechanism involving Asp-151 and Lys-148 with Lys-148, Arg-326, Lys-315, and perhaps Arg-186 being important for substrate binding.
Xu
J
J
Department of Biological Chemistry, Chicago Medical School, Illinois 60064.
Green
P C
PC
Kemp
R G
RG
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D011756:Diphosphates / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010770:Phosphotransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011424:Propionibacterium / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011756:Diphosphates; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001120:Arginine; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase