Amino acid sequence at the citrate allosteric site of rabbit muscle phosphofructokinase.
Previously, this laboratory has demonstrated [Colombo, G., & Kemp, R. G. (1976) Biochemistry 15, 1774-1780] that under appropriate conditions the citrate inhibitory binding site of rabbit skeletal muscle phosphofructokinase can be covalently modified by using pyridoxal phosphate and sodium borohydride. In the current study, phosphofructokinase was modified by [3H]pyridoxal phosphate and sodium borohydride with or without the addition of citrate to protect the ligand binding site. The modified proteins were digested with trypsin, and the peptides were separated by high-pressure liquid chromatography. A comparison of the tryptic chromatographic profiles showed that while the label was broadly distributed among nine peaks in the elution profile of the enzyme modified in the presence of the protective ligand, a single peptide contained 70% of the total radioactivity of the enzyme modified in the absence of citrate. This peptide was presumed to contain at least part of the citrate inhibitory site of the enzyme. The sequence of the peptide was determined and shown to match with positions 528-536 of phosphofructokinase with the modified residue being Lys-529. A comparison of the sequence with that of procaryotic phosphofructokinase indicated that a homologous residue in the enzyme from Bacillus stearothermophilis is critical to an allosteric site. A second peptide that was the most abundant labeled peptide in the digest of the enzyme modified in the presence of citrate was found to be identical with the second most abundant peptide of the digest from the unprotected enzyme. This peptide corresponded to residues 681-692 with the lysine at position 684 being the site of phosphopyridoxylation.(ABSTRACT TRUNCATED AT 250 WORDS)
D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D002951:Citrates / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011817:Rabbits; D014357:Trypsin
D002951:Citrates; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D010732:Phosphofructokinase-1; D014357:Trypsin