Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence.
Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.
Kettlun
A M
AM
Departamento de Bioquímica y Biologia Molecular, Universidade de Chile, Santiago, Chile.
Espinosa
V
V
Zanocco
A
A
Valenzuela
M A
MA
D000244:Adenosine Diphosphate / Q000378:metabolism*; D001081:Apyrase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D009711:Nucleotides / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011198:Solanum tuberosum / Q000737:chemistry / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D007527:Isoenzymes; D009711:Nucleotides; D010940:Plant Proteins; D000244:Adenosine Diphosphate; D001081:Apyrase