Site-specific effects of zinc on the activity of family II pyrophosphatase

Family II pyrophosphatases (PPases), recently found in bacteria and archaebacteria, are Mn2+ -containing metalloenzymes with two metal-binding subsites (M1 and M2) in the active site. These PPases can use a number of other divalent metal ions as the cofactor but are inactive with Zn2+, which is known to be a good cofactor for family I PPases. We report here that the Mg2+-bound form of the family II PPase from Streptococcus gordonii is nearly instantly activated by incubation with equimolar Zn2+, but the activity thereafter decays on a time scale of minutes. The activation of the Mn2+-form by Zn2+ was slower but persisted for hours, whereas activation was not observed with the Ca2+- and apo-forms. The bound Zn2+ could be removed from PPase by prolonged EDTA treatment, with a complete recovery of activity. On the basis of the effect of Zn2+ on PPase dimerization, the Zn2+ binding constant appeared to be as low as 10(-12) M for S. gordonii PPase. Similar effects of Zn2+ and EDTA were observed with the Mg2+- and apo-forms of Streptococcus mutans and Bacillus subtilis PPases. The effects of Zn2+ on the apo- and Mg2+-forms of HQ97 and DE15 B. subtilis PPase variants (modified M2 subsite) but not of HQ9 variant (modified M1 subsite) were similar to that for the Mn2+-form of wild-type PPase. These findings can be explained by assuming that (a) the PPase tightly binds Mg2+ and Mn2+ at the M2 subsite; (b) the activation of the corresponding holoenzymes by Zn2+ results from its binding to the MI subsite; and (c) the subsequent inactivation of Mg2+-PPase results from Zn2+ migration to the M2 subsite. The inability of Zn2+ to activate apo-PPase suggests that Zn2+ binds more tightly to M2 than to MI, allowing direct binding to M2. Zn2+ is thus an efficient cofactor at subsite MI but not at subsite M2.