Brain pyridoxal kinase: photoaffinity labeling of the substrate-binding site.
4-Benzoylbenzoic acid inhibits pyridoxal kinase activity competitively with respect to pyridoxal. The Ki was determined to be 5 x 10(-5) M. Binding studies showed that 4-benzoylbenzoic acid bound to pyridoxal kinase at a 1:1 molar ratio and with a dissociation constant (Kd) of 5.9 x 10(-5) M. Photoirradiation of pyridoxal kinase in the presence of a 10-fold excess of 4-benzoylbenzoic acid at pH 6.5 resulted in an irreversible loss of enzymatic activity; this photoinactivation was prevented by the presence of pyridoxal. Amino acid analysis revealed that 1 tyrosine residue/subunit was modified during photoinactivation. The presence of a tyrosine residue at the active site of pyridoxal kinase was confirmed by reaction with tetranitromethane. In the presence of 1 x 10(-4) M tetranitromethane, a complete loss of the kinase activity was observed after incubation at 25 degrees C for 8 min, with modification of a total of 3 tyrosine residues. The second-order rate constant (K2) of the reaction between the tyrosine residues and tetranitromethane was determined to be 53.3 s-1 M-1.
Scholz
G
G
School of Pharmacy, South Australian Institute of Technology, Adelaide.
Kwok
F
F
D000345:Affinity Labels / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001577:Benzophenones / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010770:Phosphotransferases / Q000378:metabolism*; D010777:Photochemistry; D011730:Pyridoxal / Q000378:metabolism; D011731:Pyridoxal Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012756:Sheep; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D000345:Affinity Labels; D000596:Amino Acids; D001577:Benzophenones; D011730:Pyridoxal; D014443:Tyrosine; C058818:4-benzoylbenzoic acid; D010770:Phosphotransferases; D011731:Pyridoxal Kinase; D013774:Tetranitromethane