Site-specific effects of zinc on the activity of family II pyrophosphatase.
Family II pyrophosphatases (PPases), recently found in bacteria and archaebacteria, are Mn(2+)-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 Zn(2+), which is known to be a good cofactor for family I PPases. We report here that the Mg(2+)-bound form of the family II PPase from Streptococcus gordonii is nearly instantly activated by incubation with equimolar Zn(2+), but the activity thereafter decays on a time scale of minutes. The activation of the Mn(2+)-form by Zn(2+) was slower but persisted for hours, whereas activation was not observed with the Ca(2+)- and apo-forms. The bound Zn(2+) could be removed from PPase by prolonged EDTA treatment, with a complete recovery of activity. On the basis of the effect of Zn(2+) on PPase dimerization, the Zn(2+) binding constant appeared to be as low as 10(-12) M for S. gordonii PPase. Similar effects of Zn(2+) and EDTA were observed with the Mg(2+)- and apo-forms of Streptococcus mutans and Bacillus subtilis PPases. The effects of Zn(2+) on the apo- and Mg(2+)-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 Mn(2+)-form of wild-type PPase. These findings can be explained by assuming that (a) the PPase tightly binds Mg(2+) and Mn(2+) at the M2 subsite; (b) the activation of the corresponding holoenzymes by Zn(2+) results from its binding to the M1 subsite; and (c) the subsequent inactivation of Mg(2+)-PPase results from Zn(2+) migration to the M2 subsite. The inability of Zn(2+) to activate apo-PPase suggests that Zn(2+) binds more tightly to M2 than to M1, allowing direct binding to M2. Zn(2+) is thus an efficient cofactor at subsite M1 but not at subsite M2.
Zyryanov
Anton B
AB
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia.
Tammenkoski
Marko
M
Salminen
Anu
A
Kolomiytseva
Galina Ya
GY
Fabrichniy
Igor P
IP
Goldman
Adrian
A
Lahti
Reijo
R
Baykov
Alexander A
AA
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D004492:Edetic Acid / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D004793:Enzyme Reactivators / Q000737:chemistry; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D013295:Streptococcus mutans / Q000201:enzymology / Q000235:genetics; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D004793:Enzyme Reactivators; D008667:Metalloproteins; D008345:Manganese; D004492:Edetic Acid; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D015032:Zinc; D002118:Calcium