Espinosa V, Kettlun A M, Zanocco A, Cardemil E, Valenzuela M A
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile.
Phytochemistry. 2003 May;63(1):7-14. doi: 10.1016/s0031-9422(02)00672-6.
Comparative studies of intrinsic and extrinsic fluorescence of apyrases purified from two potato tuber varieties (Pimpernel and Desirée) were performed to determine differences in the microenvironment of the nucleotide binding site. The dissociation constants (K(d)) of Pimpernel apyrase for the binding of different fluorescent substrate analogs: methylanthranoyl (MANT-), trinitrophenyl (TNP-), and epsilon -derivatives of ATP and ADP were determined from the quenching of Trp fluorescence, and compared with K(d) values previously reported for Desirée enzyme. Binding of non-fluorescent substrate analogues decreased the Trp emission of both isoapyrases, indicating conformational changes in the vicinity of these residues. Similar effect was observed with fluorescent derivatives where, in the quenching effect, the transfer of energy from tryptophan residues to the fluorophore moiety could be additionally involved. The existence of energy transfer between Trp residues in the Pimpernel enzyme was demonstrated with epsilon -analogues, similar to our previous observations with the Desirée. From these results we deduced that tryptophan residues are close to or in the nucleotide binding site in both enzymes. Experiments with quenchers like acrylamide, Cs(+) and I(-), both in the presence and absence of nucleotide analogues, suggest the existence of differences in the nucleotide binding site of the two enzymes. From the results obtained in this work, we can conclude that the differences found in the microenvironment of the nucleotide binding site can explain, at least in part, the kinetic behaviour of both isoenzymes.
对从两个马铃薯品种（小繁缕和德西蕾）中纯化的腺苷三磷酸双磷酸酶的内在荧光和外在荧光进行了比较研究，以确定核苷酸结合位点微环境的差异。通过色氨酸荧光猝灭测定了小繁缕腺苷三磷酸双磷酸酶对不同荧光底物类似物（甲基蒽甲酰基（MANT-）、三硝基苯基（TNP-）以及ATP和ADP的ε-衍生物）结合的解离常数（K(d)），并与先前报道的德西蕾酶的K(d)值进行了比较。非荧光底物类似物的结合降低了两种同工腺苷三磷酸双磷酸酶的色氨酸发射，表明这些残基附近发生了构象变化。在荧光衍生物中也观察到了类似的效应，在猝灭效应中，可能还涉及从色氨酸残基到荧光团部分的能量转移。用ε-类似物证明了小繁缕酶中色氨酸残基之间存在能量转移，这与我们之前对德西蕾酶的观察结果相似。从这些结果我们推断，两种酶中的色氨酸残基都靠近或位于核苷酸结合位点。在有和没有核苷酸类似物存在的情况下，用丙烯酰胺、Cs(+)和I(-)等猝灭剂进行的实验表明，两种酶的核苷酸结合位点存在差异。从这项工作中获得的结果可以得出结论，在核苷酸结合位点微环境中发现的差异至少可以部分解释两种同工酶的动力学行为。
