Covalent modification of the solubilized rat liver vitamin K-dependent carboxylase with pyridoxal-5'-phosphate.
The carboxylation of the pentapeptide substrate, Phe-Leu-Glu-Glu-Ile, by a rat microsomal vitamin K-dependent carboxylase was stimulated two- to threefold at pyridoxal-5'-P concentrations between 0.5 and 1.0 mM. This stimulation was reduced at concentrations higher than 1.0 mM. The Km for the pentapeptide was lowered twofold in the presence of 1 mM pyridoxal-5'-P. The activation by pyridoxal-5'-P is specific, as 1 mM pyridoxal, pyridoxine, pyridoxine-5'-P, pyridoxamine, pyridoxamine-5'-P, or 4-pyridoxic acid did not stimulate the pentapeptide carboxylation rate. All six analogs, as well as formaldehyde and acetaldehyde, inhibited the carboxylation reaction in a concentration-dependent manner. The activation of the carboxylase by pyridoxal-5'-P appeared to be mediated by its direct binding to the enzyme via Schiff base formation. Sodium borohydride reduction of solubilized microsomes in the presence of pyridoxal-5'-P, followed by dialysis to remove unbound material, resulted in a carboxylase preparation with a specific activity twice that of the untreated control microsomes. The derivatized enzyme was not further stimulated by added pyridoxal-5'-P. This derivatized carboxylase could be obtained in the absence of pentapeptide and divalent cations. The stimulation of the carboxylase activity by divalent cations and pyridoxal-5'-P was mediated at separate site(s) on the enzyme. Studies of the NH2-terminal pyridoxalated pentapeptide with both a normal and PLP-modified enzyme, in the presence and absence of PLP, demonstrated competition of the pentapeptide PLP moiety to a PLP site on the enzyme. It was concluded that pyridoxal-5'-P forms a covalent attachment to an epsilon-NH2 of a lysine near the active site of the carboxylase.
D000818:Animals; D019735:Carbon-Carbon Ligases; D055598:Chemical Phenomena; D002621:Chemistry; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D008025:Ligases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D051381:Rats; D012995:Solubility
D009842:Oligopeptides; C040586:phenylalanyl-leucyl-glutamyl-glutamyl-isoleucine; D011732:Pyridoxal Phosphate; D004492:Edetic Acid; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase; D008274:Magnesium