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.
Phytochemistry. 2000 Aug;54(8):995-1001. doi: 10.1016/s0031-9422(99)00528-2.
Chemical modification of potato apyrase suggests that tryptophan residues are close to the nucleotide binding site. Kd values (+/- Ca2+) for the complexes of apyrase with the non-hydrolysable phosphonate adenine nucleotide analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(alpha,beta-methylene) diphosphate, were obtained from quenching of the intrinsic enzyme fluorescence. Other fluorescent nucleotide analogues (2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-diphosphate. 1,N6-ethenoadenosine triphosphate and 1,N6-ethenoadenosine diphosphate) were hydrolysed by apyrase in the presence of Ca2+, indicating binding to the active site. The dissociation constants for the binding of these analogues were calculated from both the decrease of the protein (tryptophan) fluorescence and enhancement of the nucleotide fluorescence. Using the sensitised acceptor (nucleotide analogue) fluorescence method, energy transfer was observed between enzyme tryptophans and ethene-derivatives. These results support the view that tryptophan residues are present in the nucleotide-binding region of the protein, appropriately oriented to allow the energy transfer process to occur.
马铃薯三磷酸腺苷双磷酸酶的化学修饰表明，色氨酸残基靠近核苷酸结合位点。通过淬灭内在酶荧光，获得了三磷酸腺苷双磷酸酶与不可水解的膦酸酯腺嘌呤核苷酸类似物腺苷5'-（β，γ-亚甲基）三磷酸和腺苷5'-（α，β-亚甲基）二磷酸形成的复合物的Kd值（±Ca2+）。其他荧光核苷酸类似物（2'（3'）-O-（2,4,6-三硝基苯基）腺苷5'-三磷酸、2'（3'）-O-（2,4,6-三硝基苯基）腺苷5'-二磷酸、1,N6-乙烯腺苷三磷酸和1,N6-乙烯腺苷二磷酸）在Ca2+存在下被三磷酸腺苷双磷酸酶水解，表明它们与活性位点结合。这些类似物结合的解离常数通过蛋白质（色氨酸）荧光的降低和核苷酸荧光的增强来计算。使用敏化受体（核苷酸类似物）荧光法，观察到酶色氨酸与乙烯衍生物之间的能量转移。这些结果支持这样一种观点，即色氨酸残基存在于蛋白质的核苷酸结合区域，其取向适当，能够发生能量转移过程。
