Site-Specific Effects of Zinc on the Activity of Family II Pyrophosphatase

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• 作者：Anton B. Zyryanov ; Marko Tammenkoski ; Anu Salminen ; Galina Ya. Kolomiytseva ; Igor P. Fabrichniy ; Adrian Goldman ; Reijo Lahti ; and Alexander A. Baykov
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：November 16, 2004
• 年：2004
• 卷：43
• 期：45
• 页码：14395 - 14402
• 全文大小：97K
• 年卷期：v.43,no.45(November 16, 2004)
• ISSN：1520-4995

文摘

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 PPasescan use a number of other divalent metal ions as the cofactor but are inactive with Zn²⁺, which is knownto be a good cofactor for family I PPases. We report here that the Mg²⁺-bound form of the family IIPPase from Streptococcus gordonii is nearly instantly activated by incubation with equimolar Zn²⁺, butthe activity thereafter decays on a time scale of minutes. The activation of the Mn²⁺-form by Zn²⁺ wasslower but persisted for hours, whereas activation was not observed with the Ca²⁺- and apo-forms. Thebound Zn²⁺ could be removed from PPase by prolonged EDTA treatment, with a complete recovery ofactivity. On the basis of the effect of Zn²⁺ on PPase dimerization, the Zn²⁺ binding constant appeared tobe as low as 10^-12 M for S. gordonii PPase. Similar effects of Zn²⁺ and EDTA were observed with theMg²⁺- and apo-forms of Streptococcus mutans and Bacillus subtilis PPases. The effects of Zn²⁺ on theapo- and Mg²⁺-forms of HQ97 and DE15 B. subtilis PPase variants (modified M2 subsite) but not ofHQ9 variant (modified M1 subsite) were similar to that for the Mn²⁺-form of wild-type PPase. Thesefindings 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 M1 subsite;and (c) the subsequent inactivation of Mg²⁺-PPase results from Zn²⁺ migration to the M2 subsite. Theinability of Zn²⁺ to activate apo-PPase suggests that Zn²⁺ binds more tightly to M2 than to M1, allowingdirect binding to M2. Zn²⁺ is thus an efficient cofactor at subsite M1 but not at subsite M2.