Kwok F, Churchich J E
Department of Biochemistry, University of Tennessee, Knoxville 37916.
Eur J Biochem. 1991 Jul 1;199(1):157-62. doi: 10.1111/j.1432-1033.1991.tb16104.x.
Fluorescence spectroscopy, static and dynamic, was applied to deduce proximity relationships between paramagnetic metal ions chelated to ATP and the inhibitor pyridoxal oxime bound to the site of the substrate pyridoxal. The fluorescence yield of free pyridoxal oxime is considerably reduced in the presence of CoCl2 (1 mM) due to the formation of a non-fluorescent complex. The fluorescence properties of pyridoxal oxime bound to pyridoxal kinase (50 microM, Kd = 1 microM) remain invariant in the presence of CoCl2 (1 mM) and ATP (1 mM). The results are interpreted to mean that the distances are too great for direct or first-coordination-sphere interaction between pyridoxaloxime and Co(II) centers. The bidentate complex Cr(III).ATP is a competitive inhibitor of ATP (Ki = 83 microM) and its binding to pyridoxal kinase (50 microM) complexed to pyridoxal oxime (50 microM) was monitored by static and dynamic fluorescence spectroscopy. The fluorescence yield of bound pyridoxal oxime is reduced by 25% in the presence of Cr(III).ATP (0.8 mM) leading to a decrease in fluorescence lifetime from (tau) = 6.60 ns to (tau) = 4.20 ns. The decrease in fluorescence yield together with a diminution in fluorescence lifetime can be explained in terms of radiationless energy transfer from excited pyridoxal oxime to Cr(III) chelated to ATP. Using Forster's equation and an efficiency of energy transfer of 0.25, it was found that a distance of 1.3 nm separates pyridoxal oxime from the Cr(III) center.
运用静态和动态荧光光谱法，来推断与ATP螯合的顺磁性金属离子和结合于底物吡哆醛位点的抑制剂吡哆醛肟之间的邻近关系。由于形成了非荧光复合物，在CoCl2（1 mM）存在的情况下，游离吡哆醛肟的荧光产率显著降低。结合于吡哆醛激酶（50 microM，Kd = 1 microM）的吡哆醛肟的荧光特性，在CoCl2（1 mM）和ATP（1 mM）存在时保持不变。这些结果被解释为意味着吡哆醛肟与Co（II）中心之间的距离太大，无法发生直接或第一配位球相互作用。双齿复合物Cr（III）.ATP是ATP的竞争性抑制剂（Ki = 83 microM），通过静态和动态荧光光谱法监测其与结合了吡哆醛肟（50 microM）的吡哆醛激酶（50 microM）的结合情况。在Cr（III）.ATP（0.8 mM）存在的情况下，结合的吡哆醛肟的荧光产率降低了25%，导致荧光寿命从（tau） = 6.60 ns降至（tau） = 4.20 ns。荧光产率的降低以及荧光寿命的缩短，可以用从激发态的吡哆醛肟到与ATP螯合的Cr（III）的无辐射能量转移来解释。使用福斯特方程和0.25的能量转移效率，发现吡哆醛肟与Cr（III）中心之间的距离为1.3 nm。
