Effect of modification of lysine residues of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase with pyridoxal 5'-phosphate.
Inactivation of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase by pyridoxal 5'-P followed by reduction with NaBH4 was studied. Fructose-6-P,2-kinase is over 80% inactivated by 2 mM pyridoxal 5'-P. The stoichiometry of the pyridoxyl-P incorporation and the inactivation of the kinase follows a biphasic curve. The first P-pyridoxyl residue incorporated per protomer does not affect fructose-6-P,2-kinase, but the next two P-pyridoxyl incorporation/protomer results in 80% inactivation. The Km values for ATP and fructose-6-P of the enzymes containing varying amounts of P-pyridoxyl groups at intermediate levels of inactivation are not altered, but Vmax is decreased. Among the metabolites tested, only fructose-2,6-P2 and Mg-ATP are competitive with pyridoxal-P and protect the enzyme against the inactivation. Neither the activity nor the fructose-6-P inhibition of fructose-2,6-bisphosphatase is affected by the modification. The acid hydrolysate of the inactive P-[3H]pyridoxyl enzyme contained only [3H]pyridoxyl lysine. High performance liquid chromatography of tryptic peptides of phospho[3H]pyridoxyl enzymes reveals two peptides which were missing in the enzyme protected by fructose-2,6-P2 or ATP during the modification reaction. These peptides have been isolated, and their amino acid sequences have been determined as Asp-Gln-Asp-Lys-Tyr-Arg and Asp-Val-His-Lys-Tyr. Pyridoxal-P reacts specifically with two lysine residues at the fructose-2,6-P2-binding site of fructose-6-P,2-kinase but not that of fructose-2,6-bisphosphatase. The site may also overlap with the ATP-binding site.
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001894:Borohydrides; D055598:Chemical Phenomena; D002621:Chemistry; D005635:Fructosediphosphates / Q000494:pharmacology; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008239:Lysine; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D051381:Rats; D014357:Trypsin
D001894:Borohydrides; D005635:Fructosediphosphates; D005636:Fructosephosphates; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; C025364:sodium borohydride; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin; D008239:Lysine