Brain pyridoxal kinase dissociation of the dimeric structure and catalytic activity of the monomeric species.
Reversible dissociation of the dimeric structure of brain pyridoxal kinase into subunits was attained by addition of guanidinium HCl (2 M). The molecular mass of the subunits (40 kDa) was determined by HPLC chromatography. Separation of the processes of refolding and association of the monomeric species was achieved by attaching the protein subunits to a rigid matrix (Affi-gel 15). The matrix-bound monomer is catalytically competent. The reaction of the crosslinking reagent 4,4'-dimaleimidestilbene 2,2'-disulfonate (DMDS), a derivatized stilbene, with the dimeric structure of pyridoxal kinase resulted in the formation of an oligomeric species of 80 kDa detectable by SDS-PAGE. The crosslinked subunits exhibit the same catalytic parameters as the native enzyme. The presence of two nucleotide-binding sites per dimer was determined by fluorimetric titrations using pyridoxyl-ATP, a strong competitive inhibitor with respect to ATP. The ATP analog binds with a Kd = 5 microM to each nucleotide site of the dimeric enzyme. The mode of binding pyridoxyl-ATP to the kinase is discussed in reference to a model which assumes the presence of two binding domains per subunit.
Kwok
F
F
School of Pharmacy, South Australian Institute of Technology, Adelaide.
Scholz
G
G
Churchich
J E
JE
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines; D010770:Phosphotransferases / Q000302:isolation & purification*; D011489:Protein Denaturation; D011730:Pyridoxal / Q000494:pharmacology; D011731:Pyridoxal Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D012756:Sheep; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D006146:Guanidines; D013438:Sulfhydryl Compounds; D011730:Pyridoxal; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D011731:Pyridoxal Kinase; D019791:Guanidine