Kwok F, Churchich J E
Eur J Biochem. 1979 Jan 15;93(2):229-35. doi: 10.1111/j.1432-1033.1979.tb12815.x.
Pyridoxal kinase has been purified 2000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The interactions of the substrate pyridoxal and the inhibitor N-dansyl-2-oxopyrrolidine (dansyl = 5-dimethylaminonaphthalene-1-sulfonyl) with the catalytic site were examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase was used to determine the equilibrium dissociation constant. Pyridoxal kinase binds one molecule of substrate with a Kd = 11 microns at pH 6. The emission anisotropy spectrum of bound pyridoxal reveals that the substrate is not rigidly trapped by the protein matrix. N-Dansyl-2-oxopyrrolidine is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6 microns. N-Dansyl-2-oxopyrrolidine is immobilized by strong interactions with the enzyme, but it is displaced from the catalytic site by ATP. The results are consistent with the hypothesis that N-dansyl-2-oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.
吡哆醛激酶已从猪脑中纯化了2000倍。该酶制剂在分析凝胶电泳上以单一蛋白质和活性条带形式迁移。通过荧光光谱法研究了底物吡哆醛和抑制剂N-丹磺酰基-2-氧代吡咯烷（丹磺酰基=5-二甲基氨基萘-1-磺酰基）与催化位点的相互作用。利用吡哆醛与激酶结合后发射各向异性的增加来测定平衡解离常数。在pH 6时，吡哆醛激酶以Kd = 11微米的亲和力结合一分子底物。结合态吡哆醛的发射各向异性光谱表明，底物并未被蛋白质基质严格束缚。在吡哆醛饱和浓度下，N-丹磺酰基-2-氧代吡咯烷是ATP的竞争性抑制剂。它以6微米的解离常数与酶结合。N-丹磺酰基-2-氧代吡咯烷通过与酶的强相互作用而固定，但它会被ATP从催化位点取代。这些结果与N-丹磺酰基-2-氧代吡咯烷结合在吡哆醛激酶的核苷酸结合位点这一假设一致。
