Binding of a photoaffinity analogue of pyridoxal to pyridoxal kinase.
The binding of pyridoxal analogues to the structural domains of pyridoxal kinase was studied by fluorescence spectroscopy and chromatographic techniques. Two fragments of 24 and 16 kDa, arising from limited proteolysis of the native enzyme, were separated by ion-exchange chromatography and used for binding studies with pyridoxal oxime. Fluorometric titrations yielded dissociation constants of 6 and 12.4 MicroM for pyridoxal oxime bound to the native enzyme and 24-kDa fragment, respectively. 4-(4-Azido-2-nitrophenyl)-pyridoxamine, a new photolabeling reagent, binds irreversibly to the kinase with concomitant loss of catalytic activity. The modified kinase (2.1 mol label/mol dimer) yields two fragments upon limited proteolysis with chymotrypsin. The two fragments were separated by reverse-phase HPLC and SDS/polyacrylamide gel electrophoresis. Radiolabeled ligand was detected only in the 24-kDa fragment. It is postulated that the pyridoxal binding site is located in the 24-kDa structural domain.
Scholz
G
G
Department of Biochemistry, University of Tennessee, Knoxville 37996-8040.
Kwok
F
F
Churchich
J E
JE
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D010091:Oximes; D011730:Pyridoxal / Q000031:analogs & derivatives / Q000378:metabolism*; D011731:Pyridoxal Kinase / Q000378:metabolism*; D011733:Pyridoxamine / Q000031:analogs & derivatives; D012756:Sheep; D013050:Spectrometry, Fluorescence
D000345:Affinity Labels; D001386:Azides; D004791:Enzyme Inhibitors; D010091:Oximes; C066355:4-(4-azido-2-nitrophenyl)pyridoxamine; D011730:Pyridoxal; D011733:Pyridoxamine; D011731:Pyridoxal Kinase; D002918:Chymotrypsin