Proteolytic cleavage of pyridoxal kinase into two structural domains.
Chymotryptic digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields 2 fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were separated by chromatographic techniques and analyzed for interaction with the ATP analogue, trinitrophenyl-ATP, using fluorescence spectroscopy. The absorption and fluorescence properties of trinitrophenyl-ATP bound to the fragment of 24 kDa (emission maximum, 540 nm, emission anisotropy at 460 nm, 0.30, and fluorescence lifetime, gamma = lns) are indistinguishable from those of the ATP analogue bound to the native enzyme. The fragment of 16 kDa does not bind trinitrophenyl-ATP. The results are consistent with the hypothesis that monomeric pyridoxal kinase is folded into 2 domains connected by a single polypeptide chain sensitive to proteolytic cleavage.
Dominici
P
P
Department of Biochemistry, University of Tennessee, Knoxville.
Kwok
F
F
Churchich
J E
JE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D010446:Peptide Fragments / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011731:Pyridoxal Kinase / Q000378:metabolism*; D012756:Sheep; D013057:Spectrum Analysis
D000596:Amino Acids; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D011731:Pyridoxal Kinase; D002918:Chymotrypsin