Sheep brain pyridoxal kinase: fluorescence spectroscopy of the dimeric enzyme.
Pyridoxal kinase (ATP:pyridoxal 5-phosphotransferase, EC 2.7.1.35) has been purified 9000-fold from sheep brain by affinity chromatography. The enzyme of 80,000 molecular weight is made up of two identical-size subunits. The interaction of the inhibitor N-dansyl-1,8-diaminooctane with the nucleotide site of the kinase was examined by means of steady and nanosecond fluorescence spectroscopy. N-Dansyl-1,8-diaminooctane is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the nucleotide site of the enzyme with Kd = 2.2 microM. Bound N-dansyl-1,8-diaminooctane is shielded from collisional encounters with the external quencher acrylamide. The collisional rate constant for bound N-dansyl-1,8-diaminooctane (Kq = 1.4 X 10(8) M-1 X s-1) is 10-times lower than the value obtained for the free chromophore. Nanosecond emission anisotropy measurements yield a rotational correlation time of 42 ns for the inhibitor complexes to the kinase. Both steady and nanosecond fluorescence results are consistent with a model in which the inhibitor bound to the nucleotide site is immobilized by amino acids located at the catalytic site.
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D003619:Dansyl Compounds / Q000494:pharmacology*; D003959:Diamines / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010770:Phosphotransferases / Q000378:metabolism*; D011731:Pyridoxal Kinase / Q000302:isolation & purification / Q000378:metabolism*; D012756:Sheep; D013050:Spectrometry, Fluorescence / Q000379:methods
D003619:Dansyl Compounds; D003959:Diamines; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; C050794:N-dansyl-1,8-diaminooctane; D010770:Phosphotransferases; D011731:Pyridoxal Kinase