The interaction of paramagnetic ions chelated to ATP with pyridoxal analogues. Fluorescence studies of pyridoxal kinase.
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.
Kwok
F
F
Department of Biochemistry, University of Tennessee, Knoxville 37916.
Churchich
J E
JE
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002614:Chelating Agents; D007477:Ions; D008280:Magnetics; D011730:Pyridoxal / Q000031:analogs & derivatives / Q000737:chemistry; D011731:Pyridoxal Kinase / Q000378:metabolism*; D012756:Sheep; D013050:Spectrometry, Fluorescence
D002614:Chelating Agents; D007477:Ions; D011730:Pyridoxal; C028073:pyridoxaloxime; D000255:Adenosine Triphosphate; D011731:Pyridoxal Kinase