Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogenei ty from several clonal varieties of Solanum tuberosum. Depending on the sou rce of the enzyme, differences in kinetic and physicochemical properties ha ve been described, which cannot be explained by the amino acid residues pre sent in the active site. In order to understand the different kinetic behav ior of the Pimpernel (ATPase/ADPase = 10) and Desiree (ATPase/ADPase = 1) i soenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan, The intrinsic fluorescence of th e two apyrases was slightly different. The maximum emission wavelengths of the Desiree and Pimpernel enzymes were 336 and 340 nm, respectively, sugges ting small differences in the microenvironment of Trp residues. The Pimpern el enzyme emitted more fluorescence than the Desiree apyrase at the same co ncentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescenc e emission of both apyrases, indicating the presence of conformational chan ges in the neighborhood of Trp residues. Experiments with quenchers of diff erent 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. Differenc es in the nucleotide-binding site may explain, at least in part, the kineti c differences of the Pimpernel and Desiree isoapyrases.