PURIFICATION AND CHARACTERIZATION OF AN EXOPOLYPHOSPHATASE FROM SACCHAROMYCES-CEREVISIAE

An exopolyphosphatase (polyphosphate phosphohydrolase; EC 3.6.1.11) ac tivity that cleaves inorganic polyphosphates to orthophosphate has bee n purified to apparent homogeneity (>95% pure) from Saccharomyces cere visiae, The exopolyphosphatase is a monomeric protein with a polypepti de molecular mass of 28 kDa. The enzyme, which can be stabilized in th e presence of Triton X-100, has a pH optimum of 7.5 and requires, for maximal activity, Co2+ or Mg2+ ions. In the absence of these ions, the exopolyphosphatase binds to polyphosphate but does not degrade it, al lowing affinity purification of the enzyme on a polyphosphate-modified zirconia support. o-Vanadate, Cu2+, and Ca2+ are effective inhibitors of the exopolyphosphatase. The enzyme preferentially hydrolyzes linea r polyphosphates in a nonprocessive manner; pyrophosphate as well as c yclic tri- and tetrametaphosphate are degraded only at very low rates, whereas ATP is not split by the exopolyphosphatase. The only product formed by the action of the enzyme is orthophosphate.