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Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
Department of Biochemistry, University of Turku, Turku, Finland.
Center for Ulcer Research and Education, Department of Medicine, University of California, Los Angeles, California, USA.
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2000 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 45, 13931-13938 p.Article in journal (Refereed) Published
Abstract [en]

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2000. Vol. 39, no 45, 13931-13938 p.
National Category
Cell Biology
URN: urn:nbn:se:liu:diva-132130DOI: 10.1021/bi000895sISI: 000165412800036PubMedID: 11076535ScopusID: 2-s2.0-0034649468OAI: diva2:1037772
Available from: 2016-10-18 Created: 2016-10-18 Last updated: 2016-12-06Bibliographically approved

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