Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate.
The pyruvate dehydrogenase multienzyme complex from bovine kidney and heart is inactivated by treatment with pyridoxal 5'-phosphate and sodium cyanide or sodium borohydride. The site of this inhibition is the pyruvate dehydrogenase (E1) component of the complex. Inactivation of E1 by the pyridoxal phosphate-cyanide treatment was prevented by thiamin pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamin pyrophosphate binding sites per molecule (alpha 2 beta 2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 5-fold. Incorporation of approximately 2.4 equiv of 14CN per mole of E1 tetramer in the presence of pyridoxal phosphate resulted in about a 90% loss of E1 activity. Radioactivity was incorporated predominantly into the E1 alpha subunit. Radioactive N6-pyridoxyllysine was identified in an acid hydrolysate of the E1-pyridoxal phosphate complex that had been reduced with NaB3H4. The data are interpreted to indicate that in the presence of sodium cyanide or sodium borohydride, pyridoxal phosphate reacts with a lysine residue at or near the thiamin pyrophosphate binding site of E1. This binding site is apparently located on the alpha subunit.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D012966:Sodium Cyanide / Q000494:pharmacology
D011768:Pyruvate Dehydrogenase Complex; D011732:Pyridoxal Phosphate; D012966:Sodium Cyanide