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• W2006063265 abstract "A homohexameric molecule of Escherichia coli pyrophosphatase is arranged as a dimer of trimers, with an active site present in each of its six monomers. Earlier we reported that substitution of His136 and His140 in the intertrimeric subunit interface splits the molecule into active trimers (Velichko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyytiä, T., Goldman, A., Cooperman, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemistry 37, 734–740). Here we demonstrate that additional substitutions of Tyr77 and Gln80 in the intratrimeric interface give rise to moderately active dimers or virtually inactive monomers, depending on pH, temperature, and Mg2+ concentration. Successive dissociation of the hexamer into trimers, dimers, and monomers progressively decreases the catalytic efficiency (by 106-fold in total), and conversion of a trimer into dimer decreases the affinity of one of the essential Mg2+-binding sites/monomer. Disruptive substitutions predominantly in the intratrimeric interface stabilize the intertrimeric interface and vice versa, suggesting that the optimal intratrimeric interaction is not compatible with the optimal intertrimeric interaction. Because of the resulting “conformational strain,” hexameric wild-type structure appears to be preformed to bind substrate. A hexameric triple variant substituted at Tyr77, Gln80, and His136 exhibits positive cooperativity in catalysis, consistent with this model. A homohexameric molecule of Escherichia coli pyrophosphatase is arranged as a dimer of trimers, with an active site present in each of its six monomers. Earlier we reported that substitution of His136 and His140 in the intertrimeric subunit interface splits the molecule into active trimers (Velichko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyytiä, T., Goldman, A., Cooperman, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemistry 37, 734–740). Here we demonstrate that additional substitutions of Tyr77 and Gln80 in the intratrimeric interface give rise to moderately active dimers or virtually inactive monomers, depending on pH, temperature, and Mg2+ concentration. Successive dissociation of the hexamer into trimers, dimers, and monomers progressively decreases the catalytic efficiency (by 106-fold in total), and conversion of a trimer into dimer decreases the affinity of one of the essential Mg2+-binding sites/monomer. Disruptive substitutions predominantly in the intratrimeric interface stabilize the intertrimeric interface and vice versa, suggesting that the optimal intratrimeric interaction is not compatible with the optimal intertrimeric interaction. Because of the resulting “conformational strain,” hexameric wild-type structure appears to be preformed to bind substrate. A hexameric triple variant substituted at Tyr77, Gln80, and His136 exhibits positive cooperativity in catalysis, consistent with this model. inorganic pyrophosphatase E. coli PPase dimagnesium pyrophosphate wild type 4-morpholineethanesulfonic acid 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid 3-(cyclohexylamino)propanesulfonic acid 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid Inorganic pyrophosphatase (EC 3.6.1.1; PPase)1 catalyzes the interchange between pyrophosphate and orthophosphate and is essential for life (1Chen J. Brevet A. Formant M. Leveque F. Schmitter J.-M. Blanquet S. Plateau P. J. Bacteriol. 1990; 172: 5686-5689Crossref PubMed Google Scholar, 2Lundin M. Baltscheffsky H. Ronne H. J. Biol. Chem. 1991; 266: 12168-12172Abstract Full Text PDF PubMed Google Scholar). Because of its relative simplicity and high efficiency (k cat/K m = ∼109m−1 s−1), PPase has become a paradigm for mechanistic and structural studies of enzymatic phosphoryl transfer from phosphoric acid anhydrides to water. The best studied PPases are those from Escherichia coli andSaccharomyces cerevisiae (3Cooperman B.S. Baykov A.A. Lahti R. Trends Biochem. Sci. 1992; 17: 262-266Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 4Baykov A.A. Cooperman B.S. Goldman A. Lahti R. Progr. Mol. Subcell. Biol. 1999; 23: 127-150Crossref PubMed Scopus (94) Google Scholar).A molecule of E. coli PPase (E-PPase) is formed by six identical subunits, 20 kDa each, arranged with D3 symmetry in two layers of trimers (Fig.1 A). The three or four contact zones (see below), which are not well separated, cover about 24% of the accessible surface area of a monomer. The intratrimer contacts are of a circular “head-to-tail” type, meaning that each monomer has two different intratrimer contact regions (Fig. 1 B) (5Kankare J. Salminen T. Lahti R. Cooperman B. Baykov A.A. Goldman A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 551-563Crossref PubMed Scopus (36) Google Scholar,6Salminen T. Teplyakov A. Kankare J. Cooperman B.S. Lahti R. Goldman A. Protein Sci. 1996; 5: 1014-1025Crossref PubMed Scopus (82) Google Scholar). These involve a mixture of hydrophilic and hydrophobic interactions that include Tyr77 and the backbone NH of Gln80 (5Kankare J. Salminen T. Lahti R. Cooperman B. Baykov A.A. Goldman A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 551-563Crossref PubMed Scopus (36) Google Scholar); the total surface area buried per monomer is about 1300 Å2 (6Salminen T. Teplyakov A. Kankare J. Cooperman B.S. Lahti R. Goldman A. Protein Sci. 1996; 5: 1014-1025Crossref PubMed Scopus (82) Google Scholar).There are also two different interfaces between trimers. The smaller one (140 Å2) includes a Tyr77–Asn24′ hydrogen bond (primed and unprimed numbers refer to different monomers). Tyr77 acts as a link between the intratrimer interface, the minor intertrimer interface, and the major intertrimer interface. The larger intertrimer interface in E-PPase (640 Å2) chiefly involves α-helix A, including an ion-triple formed between His140, Asp143, and His136′ (5Kankare J. Salminen T. Lahti R. Cooperman B. Baykov A.A. Goldman A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 551-563Crossref PubMed Scopus (36) Google Scholar, 8Harutyunyan E.H. Oganessyan V.Yu. Oganessyan N.N. Avaeva S.M. Nazarova T.I. Vorobyeva N.N. Kurilova S.A. Huber R. Mather T. Biochemistry. 1997; 36: 7754-7760Crossref PubMed Scopus (56) Google Scholar). Replacing either His136 or His140 with Gln destabilizes the E-PPase hexamer (9Baykov A.A. Dudarenkov V.Y. Käpylä J. Salminen T. Hyytiä T. Kasho V.N. Husgafvel S. Cooperman B.S. Goldman A. Lahti R. J. Biol. Chem. 1995; 270: 30804-30812Crossref PubMed Scopus (33) Google Scholar), whereas replacing both makes trimers the dominant species in solution even at millimolar protein concentrations (10Velichko I.S. Mikalahti K. Kasho V.N. Dudarenkov V.Y. Hyytiä T. Goldman A. Cooperman B.S. Lahti R. Baykov A.A. Biochemistry. 1998; 37: 734-740Crossref PubMed Scopus (23) Google Scholar). Another important interaction occurs through Mg2+ bound at the intertrimeric interface. The Mg2+ ion is octahedrally associated with six water molecules, which in turn hydrogen bond to the side chains of Asn24/24′ and Asp26/26′, as well as to the backbone carbonyls of Asn24 and Ala25(8Harutyunyan E.H. Oganessyan V.Yu. Oganessyan N.N. Avaeva S.M. Nazarova T.I. Vorobyeva N.N. Kurilova S.A. Huber R. Mather T. Biochemistry. 1997; 36: 7754-7760Crossref PubMed Scopus (56) Google Scholar, 11Kankare J. Salminen T. Lahti R. Cooperman B. Baykov A.A. Goldman A. Biochemistry. 1996; 35: 4670-4677Crossref PubMed Scopus (64) Google Scholar). Substitution of Asp26 with Asn or Ser eliminates Mg2+ binding to the intertrimeric site and somewhat decreases hexamer stability but hardly affects catalysis (12Efimova I.S. Salminen A. Pohjanjoki P. Lapinniemi J. Magretova N.N. Cooperman B.S. Goldman A. Lahti R. Baykov A.A. J. Biol. Chem. 1999; 274: 3294-3299Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Hexamer dissociation into trimers greatly decreases the rate constant for substrate binding to enzyme but has no effect on the catalytic constant (10Velichko I.S. Mikalahti K. Kasho V.N. Dudarenkov V.Y. Hyytiä T. Goldman A. Cooperman B.S. Lahti R. Baykov A.A. Biochemistry. 1998; 37: 734-740Crossref PubMed Scopus (23) Google Scholar, 12Efimova I.S. Salminen A. Pohjanjoki P. Lapinniemi J. Magretova N.N. Cooperman B.S. Goldman A. Lahti R. Baykov A.A. J. Biol. Chem. 1999; 274: 3294-3299Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar).Here we describe the effects of substitutions of Tyr77 and Gln80, predominantly at the intratrimeric interface, on the quaternary structure and catalytic activity of E-PPase. This interface mainly consists of hydrophobic contacts between strands and contains few hydrogen bonds between monomers (Fig. 1 C). The side chains of Tyr77 and Gln80 are thus in a rather hydrophobic environment. We show that Y77D and Q80E substitutions markedly destabilize the intratrimeric contact and, in combination with substitutions at the intertrimeric contact, yield moderately active dimeric and virtually inactive monomeric E-PPase. The results of this study also shed light on the interactions between different subunit contacts in E-PPase. Inorganic pyrophosphatase (EC 3.6.1.1; PPase)1 catalyzes the interchange between pyrophosphate and orthophosphate and is essential for life (1Chen J. Brevet A. Formant M. Leveque F. Schmitter J.-M. Blanquet S. Plateau P. J. Bacteriol. 1990; 172: 5686-5689Crossref PubMed Google Scholar, 2Lundin M. Baltscheffsky H. Ronne H. J. Biol. Chem. 1991; 266: 12168-12172Abstract Full Text PDF PubMed Google Scholar). Because of its relative simplicity and high efficiency (k cat/K m = ∼109m−1 s−1), PPase has become a paradigm for mechanistic and structural studies of enzymatic phosphoryl transfer from phosphoric acid anhydrides to water. The best studied PPases are those from Escherichia coli andSaccharomyces cerevisiae (3Cooperman B.S. Baykov A.A. Lahti R. Trends Biochem. Sci. 1992; 17: 262-266Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 4Baykov A.A. Cooperman B.S. Goldman A. Lahti R. Progr. Mol. Subcell. Biol. 1999; 23: 127-150Crossref PubMed Scopus (94) Google Scholar). A molecule of E. coli PPase (E-PPase) is formed by six identical subunits, 20 kDa each, arranged with D3 symmetry in two layers of trimers (Fig.1 A). The three or four contact zones (see below), which are not well separated, cover about 24% of the accessible surface area of a monomer. The intratrimer contacts are of a circular “head-to-tail” type, meaning that each monomer has two different intratrimer contact regions (Fig. 1 B) (5Kankare J. Salminen T. Lahti R. Cooperman B. Baykov A.A. Goldman A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 551-563Crossref PubMed Scopus (36) Google Scholar,6Salminen T. Teplyakov A. Kankare J. Cooperman B.S. Lahti R. Goldman A. Protein Sci. 1996; 5: 1014-1025Crossref PubMed Scopus (82) Google Scholar). These involve a mixture of hydrophilic and hydrophobic interactions that include Tyr77 and the backbone NH of Gln80 (5Kankare J. Salminen T. Lahti R. Cooperman B. Baykov A.A. Goldman A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 551-563Crossref PubMed Scopus (36) Google Scholar); the total surface area buried per monomer is about 1300 Å2 (6Salminen T. Teplyakov A. Kankare J. Cooperman B.S. Lahti R. Goldman A. Protein Sci. 1996; 5: 1014-1025Crossref PubMed Scopus (82) Google Scholar). There are also two different interfaces between trimers. The smaller one (140 Å2) includes a Tyr77–Asn24′ hydrogen bond (primed and unprimed numbers refer to different monomers). Tyr77 acts as a link between the intratrimer interface, the minor intertrimer interface, and the major intertrimer interface. The larger intertrimer interface in E-PPase (640 Å2) chiefly involves α-helix A, including an ion-triple formed between His140, Asp143, and His136′ (5Kankare J. Salminen T. Lahti R. Cooperman B. Baykov A.A. Goldman A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 551-563Crossref PubMed Scopus (36) Google Scholar, 8Harutyunyan E.H. Oganessyan V.Yu. Oganessyan N.N. Avaeva S.M. Nazarova T.I. Vorobyeva N.N. Kurilova S.A. Huber R. Mather T. Biochemistry. 1997; 36: 7754-7760Crossref PubMed Scopus (56) Google Scholar). Replacing either His136 or His140 with Gln destabilizes the E-PPase hexamer (9Baykov A.A. Dudarenkov V.Y. Käpylä J. Salminen T. Hyytiä T. Kasho V.N. Husgafvel S. Cooperman B.S. Goldman A. Lahti R. J. Biol. Chem. 1995; 270: 30804-30812Crossref PubMed Scopus (33) Google Scholar), whereas replacing both makes trimers the dominant species in solution even at millimolar protein concentrations (10Velichko I.S. Mikalahti K. Kasho V.N. Dudarenkov V.Y. Hyytiä T. Goldman A. Cooperman B.S. Lahti R. Baykov A.A. Biochemistry. 1998; 37: 734-740Crossref PubMed Scopus (23) Google Scholar). Another important interaction occurs through Mg2+ bound at the intertrimeric interface. The Mg2+ ion is octahedrally associated with six water molecules, which in turn hydrogen bond to the side chains of Asn24/24′ and Asp26/26′, as well as to the backbone carbonyls of Asn24 and Ala25(8Harutyunyan E.H. Oganessyan V.Yu. Oganessyan N.N. Avaeva S.M. Nazarova T.I. Vorobyeva N.N. Kurilova S.A. Huber R. Mather T. Biochemistry. 1997; 36: 7754-7760Crossref PubMed Scopus (56) Google Scholar, 11Kankare J. Salminen T. Lahti R. Cooperman B. Baykov A.A. Goldman A. Biochemistry. 1996; 35: 4670-4677Crossref PubMed Scopus (64) Google Scholar). Substitution of Asp26 with Asn or Ser eliminates Mg2+ binding to the intertrimeric site and somewhat decreases hexamer stability but hardly affects catalysis (12Efimova I.S. Salminen A. Pohjanjoki P. Lapinniemi J. Magretova N.N. Cooperman B.S. Goldman A. Lahti R. Baykov A.A. J. Biol. Chem. 1999; 274: 3294-3299Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Hexamer dissociation into trimers greatly decreases the rate constant for substrate binding to enzyme but has no effect on the catalytic constant (10Velichko I.S. Mikalahti K. Kasho V.N. Dudarenkov V.Y. Hyytiä T. Goldman A. Cooperman B.S. Lahti R. Baykov A.A. Biochemistry. 1998; 37: 734-740Crossref PubMed Scopus (23) Google Scholar, 12Efimova I.S. Salminen A. Pohjanjoki P. Lapinniemi J. Magretova N.N. Cooperman B.S. Goldman A. Lahti R. Baykov A.A. J. Biol. Chem. 1999; 274: 3294-3299Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Here we describe the effects of substitutions of Tyr77 and Gln80, predominantly at the intratrimeric interface, on the quaternary structure and catalytic activity of E-PPase. This interface mainly consists of hydrophobic contacts between strands and contains few hydrogen bonds between monomers (Fig. 1 C). The side chains of Tyr77 and Gln80 are thus in a rather hydrophobic environment. We show that Y77D and Q80E substitutions markedly destabilize the intratrimeric contact and, in combination with substitutions at the intertrimeric contact, yield moderately active dimeric and virtually inactive monomeric E-PPase. The results of this study also shed light on the interactions between different subunit contacts in E-PPase. We thank P. V. Kalmykov for help in ultracentrifugation." @default.
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• W2006063265 title "Reciprocal Effects of Substitutions at the Subunit Interfaces in Hexameric Pyrophosphatase of Escherichia coli" @default.
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