Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

Abstract

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 thiamine pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamine pyrophosphate binding sites per molecule (.alpha.2.beta.2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 50-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 predominately 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.