The effect of bivalent metal ions on ATPase-ADPase activities of apyrase from Solanum tuberosum

Abstract

The purpose of this study was the elucidation of the possible importance of bivalent metal ions in controlling the activity of apyrase (ATP: diphosphohydrolase EC 3.6.1.5) purified from tubers of Solanum tuberosum cv Desirée. Similarities between the K m and Vm m values for ADP and Ca 2+ suggest that the true substrate of this enzyme is the metal ion-nueleotide complex. The association constant of the Ca-ADP complex was measured under the same conditions of pH and ionic strength as in the enzymatic assay system in order to calculate the true concentration of this complex. In contrast, [Mn(H 2O) 6] 2+ spin resonance spectroscopy (ESR) showed that apyrase binds this paramagnetic metal ion in the absence of ATP or ADP. The spectrum of [Mn(H 2O) 6] 2+ showed a transition at low field after the addition of apyrase. This result indicates that the binding of the enzyme produces a distortion in the electronic symmetry of [Mn(H 2O) 6] 2+. Apyrase binds other bivalent cations because hysteretic behaviour is observed when the enzyme is preincubated with bivalent metal ions in the absence of nucleotide; this hysteretic behaviour can be interpreted as a displacement of a ligand strongly bound to the enzyme (M 2+) by the substrate (Ca-ATP or Ca-ADP). The contents in vivo of Ca, Mg, Mn, Co, Zn, ATP and ADP were determined.