Kinetic studies of the pyridoxal kinase from pig liver: slow-binding inhibition by adenosine tetraphosphopyridoxal.
Pyridoxal kinase from pig liver has been purified 10,000-fold to apparent homogeneity. The enzyme is a dimer of subunits of Mr 32,000. The enzyme is strongly inhibited by the product pyridoxal 5'-phosphate. Liver pyridoxamine phosphate oxidase, another enzyme involved in the biosynthesis of pyridoxal 5'-phosphate, is also strongly inhibited by this compound [Wada, H., & Snell, E. E. (1961) J. Biol. Chem. 236, 2089-2095]. Thus, the biosynthesis of pyridoxal 5'-phosphate in the liver might be regulated by the product inhibition of both pyridoxamine phosphate oxidase and pyridoxal kinase. Kinetic studies revealed that the catalytic reaction of liver pyridoxal kinase follows an ordered mechanism in which pyridoxal and ATP bind to the enzyme and ADP and pyridoxal 5'-phosphate are released from the enzyme, in this order. Adenosine tetraphosphopyridoxal was found to be a slow-binding inhibitor of pyridoxal kinase. Pre-steady-state kinetics of the inhibition revealed that the inhibitor and the enzyme form an initial weak complex prior to the formation of a tighter and slowly reversing complex. The overall inhibition constant was 2.4 microM. ATP markedly protects the enzyme against time-dependent inhibition by the inhibitor, whereas another substrate pyridoxal affords no protection. By contrast, adenosine triphosphopyridoxal is not a slow-binding inhibitor of this enzyme.
Tagaya
M
M
Institute of Scientific and Industrial Research, Osaka University, Japan.
Yamano
K
K
Fukui
T
T
D000227:Adenine Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*; D011730:Pyridoxal / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D011731:Pyridoxal Kinase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D011732:Pyridoxal Phosphate / Q000032:analysis; D011734:Pyridoxaminephosphate Oxidase / Q000037:antagonists & inhibitors; D011817:Rabbits; D013552:Swine
D000227:Adenine Nucleotides; C049518:adenosine tetraphosphopyridoxal; D011730:Pyridoxal; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011734:Pyridoxaminephosphate Oxidase; D010770:Phosphotransferases; D011731:Pyridoxal Kinase