Effects of Active Site Mutations on the Metal Binding Affinity, Catalytic Competence, and Stability of the Family II Pyrophosphatase from Bacillus subtilis

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• 作者：Pasi Halonen ; Marko Tammenkoski ; Laila Niiranen ; Sauli Huopalahti ; Alexey N. Parfenyev ; Adrian Goldman ; Alexander Baykov ; and Reijo Lahti
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：March 15, 2005
• 年：2005
• 卷：44
• 期：10
• 页码：4004 - 4010
• 全文大小：192K
• 年卷期：v.44,no.10(March 15, 2005)
• ISSN：1520-4995

文摘

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria.Their primary and tertiary structures differ from those of the well-known family I PPases, although bothhave a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutatingone Glu, four His, and five Asp residues forming or close to the metal center on Mn²⁺ binding affinity,catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase).Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10⁴- to 10⁶-fold) reductions in the binding affinity for Mn²⁺. Most of the mutations decreased k_cat for MgPP_i by 2-3orders of magnitude when measured with Mn²⁺ or Mg²⁺ bound to the high-affinity subsite and Mg²⁺bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k_cat for the Mn-boundenzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K_mvalues were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations ofHis⁹⁷ and His⁹⁸ residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer,whereas most other mutations stabilized it. Mn²⁺, in sharp contrast to Mg²⁺, conferred high thermostabilityto wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. Theseresults indicate that family II PPases have a more integrated active site structure than family I PPases andare consequently more sensitive to conservative mutations.