ADENOSINE 5'-TETRAPHOSPHATE PHOSPHOHYDROLASE FROM YELLOW LUPIN SEEDS - PURIFICATION TO HOMOGENEITY AND SOME PROPERTIES

Adenosine 5'-tetraphosphate phosphohydrolase (EC 3.6.1.14) has been pu rified to homogeneity from the meal of yellow lupin (Lupinus luteus) s eeds. The enzyme is a single polypeptide chain of 25 +/- 1 kDa. It cat alyses the hydrolysis of a nucleoside 5'-tetraphosphate to a nucleosid e triphosphate and orthophosphate, and hydrolysis of tripolyphosphate but neither pyrophosphate nor tetraphosphate. A divalent cation, Mg2+, Co2+, Ni2+ or Mn2+, is required for these reactions. The pH optimum f or hydrolysis of adenosine 5'-tetraphosphate (p(4)A) is 8.2, V-max is 21 +/- 1.7 mu mol/min per mg of protein and the K-m for p(4)A is 3 +/- 0.6 mu M. At saturating p(4)A concentrations, the rate constant for t he reaction is 8.5 +/- 0.7 s(-1) [at 30 degrees C, in 50 mM Hepes/KOH (pH 8.2)/5 mM MgCl2/0.1 mM dithiothreitol]. p(4)A and guanosine 5'-tet raphosphate are hydrolysed at the same rate. Adenosine 5'-pentaphospha te (p(5)A) is degraded 1/200 as fast and is converted into ATP and two molecules of orthophosphate, which are liberated sequentially. This c ontrasts with the cleavage of p,A by the lupin diadenosine tetraphosph ate hydrolase (EC 3.6.1.17), which gives ATP and pyrophosphate. Zn2+, F- and Ca2+ ions inhibit the hydrolysis of p(4)A with I-50 values of 0 .1, 0.12 and 0.2 mM respectively.