Kwok F, Kerry J A, Churchich J E
Biochim Biophys Acta. 1986 Nov 21;874(2):167-73. doi: 10.1016/0167-4838(86)90114-7.
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.
通过亲和层析法已从羊脑中纯化出9000倍的吡哆醛激酶（ATP：吡哆醛5-磷酸转移酶，EC 2.7.1.35）。这种分子量为80,000的酶由两个大小相同的亚基组成。利用稳态和纳秒荧光光谱法研究了抑制剂N-丹磺酰基-1,8-二氨基辛烷与激酶核苷酸位点的相互作用。在吡哆醛饱和浓度下，N-丹磺酰基-1,8-二氨基辛烷是ATP的竞争性抑制剂。它以Kd = 2.2 microM的亲和力结合到酶的核苷酸位点。结合的N-丹磺酰基-1,8-二氨基辛烷免受与外部猝灭剂丙烯酰胺的碰撞。结合的N-丹磺酰基-1,8-二氨基辛烷的碰撞速率常数（Kq = 1.4×10^8 M^-1×s^-1）比游离发色团获得的值低10倍。纳秒发射各向异性测量得出抑制剂与激酶复合物的旋转相关时间为42 ns。稳态和纳秒荧光结果均与一种模型一致，即结合到核苷酸位点的抑制剂被位于催化位点的氨基酸固定。
