Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility.
Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desiree (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K(m) and k(cat) on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K(m) values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k(cat), indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5'-p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.
Kettlun
A M
AM
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Correo 1, Santiago, Chile.
Espinosa
V
V
García
L
L
Valenzuela
M A
MA
D000345:Affinity Labels; D001081:Apyrase / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D007527:Isoenzymes; D007700:Kinetics; D010940:Plant Proteins; D035281:Plant Tubers / Q000201:enzymology*; D011489:Protein Denaturation; D011198:Solanum tuberosum / Q000201:enzymology*; D013379:Substrate Specificity
D000345:Affinity Labels; D007527:Isoenzymes; D010940:Plant Proteins; D001081:Apyrase