Article

Crystal structure of holo inorganic pyrophosphatase from Escherichia coli at 1.9 Å resolution. Mechanism of hydrolysis

Lomonosov Moscow State University, Moskva, Moscow, Russia
Biochemistry (Impact Factor: 3.01). 07/1997; 36(25):7754-60. DOI: 10.1021/bi962637u
Source: PubMed

ABSTRACT Crystalline holo inorganic pyrophosphatase from Escherichia coli was grown in the presence of 250 mM MgCl2. The crystal structure has been solved by Patterson search techniques and refined to an R-factor of 17.6% at 1.9 A resolution. The upper estimate of the root-mean-square error in atomic positions is 0.26 A. These crystals belong to space group P3(2)21 with unit cell dimensions a = b = 110.27 A and c = 78.17 A. The asymmetric unit contains a trimer of subunits, i.e., half of the hexameric molecule. In the central cavity of the enzyme molecule, three Mg2+ ions, each shared by two subunits of the hexamer, are found. In the active sites of two crystallographically independent subunits, two Mg2+ ions are bound. The second active site Mg2+ ion is missing in the third subunit. A mechanism of catalysis is proposed whereby a water molecule activated by a Mg2+ ion and Tyr 55 play essential roles.

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    • "The binding site of the Mg 2ϩ ion is similar but not identical to the " tight " metal ion binding site ~M1! ~Baykov et al., 1996! in structures of E-PPase complexed with metal ion ~Arutyunyan et al., 1996; Kankare et al., 1996b; Harutyunyan et al., 1997!. In the Mg 1.5 :E-PPase complex, directly comparable to our S-PPase structure because it contains only one Mg 2ϩ ion in the active site, the Mg 2ϩ is coordinated to Asp65, Asp102 and, weakly, to Asp70; in S-PPase the Mg 2ϩ is coordinated to just two residues, Asp65 and Asp102. "
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    ABSTRACT: The first crystal structure of an inorganic pyrophosphatase (S-PPase) from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R-factor of 19.7% at 2.7 Å. S-PPase is a D3 homohexameric protein with one Mg2+ per active site in a position similar to, but not identical with, the first activating metal in mesophilic pyrophosphatases (PPase). In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg2+ while only Asp65 and Asp102 do in S-PPase, and the Mg2+ moves by 0.7 Å. S-PPase may therefore be deactivated at low temperature by mispositioning a key metal ion.The monomer S-PPase structure is very similar to that of Thermus thermophilus (T-PPase) and Escherichia coli (E-PPase), root-mean-square deviations around 1 Å/Cα. But the hexamer structures of S-and T-PPase are more tightly packed and more similar to each other than they are to that of E-PPase, as shown by the increase in surface area buried upon oligomerization. In T-PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S-PPase has hydrophilic interactions to threefold related monomers absent in both E-and T-PPase. In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E-PPase, and, especially in S-PPase, additional ionic interactions anchor the C-terminus to the rest of the protein. Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions.
    Protein Science 12/1998; 8(6):1218 - 1231. DOI:10.1110/ps.8.6.1218 · 2.85 Impact Factor
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    ABSTRACT: Inorganic pyrophosphatases (PPases, EC 3.6.1.1) hydrolyse pyrophosphate in a reaction that provides the thermodynamic 'push' for many reactions in the cell, including DNA and protein synthesis. Soluble PPases can be classified into two families that differ completely in both sequence and structure. While Family I PPases are found in all kingdoms, family II PPases occur only in certain prokaryotes. The enzyme from baker's yeast (Saccharomyces cerevisiae) is very well characterised both kinetically and structurally, but the exact mechanism has remained elusive. The enzyme uses divalent cations as cofactors; in vivo the metal is magnesium. Two metals are permanently bound to the enzyme, while two come with the substrate. The reaction cycle involves the activation of the nucleophilic oxygen and allows different pathways for product release. In this thesis I have solved the crystal structures of wild type yeast PPase and seven active site variants in the presence of the native cofactor magnesium. These structures explain the effects of the mutations and have allowed me to describe each intermediate along the catalytic pathway with a structure. Although establishing the ʻchoreographyʼ of the heavy atoms is an important step in understanding the mechanism, hydrogen atoms are crucial for the mechanism. The most unambiguous method to determine the positions of these hydrogen atoms is neutron crystallography. In order to determine the neutron structure of yeast PPase I perdeuterated the enzyme and grew large crystals of it. Since the crystals were not stable at ambient temperature, a cooling device was developed to allow neutron data collection. In order to investigate the structural changes during the reaction in real time by time-resolved crystallography a photolysable substrate precursor is needed. I synthesised a candidate molecule and characterised its photolysis kinetics, but unfortunately it is hydrolysed by both yeast and Thermotoga maritima PPases. The mechanism of Family II PPases is subtly different from Family I. The native metal cofactor is manganese instead of magnesium, but the metal activation is more complex because the metal ions that arrive with the substrate are magnesium different from those permanently bound to the enzyme. I determined the crystal structures of wild type Bacillus subtilis PPase with the inhibitor imidodiphosphate and an inactive H98Q variant with the substrate pyrophosphate. These structures revealed a new trimetal site that activates the nucleophile. I also determined that the metal ion sites were partially occupied by manganese and iron using anomalous X- ray scattering. Entsyymit ovat biologisia katalyyttejä, jotka nopeuttavat soluissa tapahtuvia kemiallisia reaktioita. Vaikka entsyymejä on tutkittu vuosikymmeniä, ovat niiden toimintamekanismit edelleen puutteellisesti ymmärrettyjä. Epäorgaaniset pyrofosfataasit ovat entsyymejä, jotka katalysoivat pyrofosfaatin pilkkoutumista kahdeksi fosfaatti-ioniksi. Tämä reaktio tuottaa käyttövoiman monille solun prosesseille, kuten proteiinien tai DNA:n synteesille. Liukoiset pyrofosfataasit voidaan jakaa kahteen perheeseen, joiden aminohappojärjestys ja rakenne ovat täysin erilaiset. Ensimmäiseen perheeseen kuuluva hiivan pyrofosfataasi on erittäin perusteellisti tutkittu entsyymi, mutta sen tarkka toimintamekanismi on pysynyt arvoituksena. Entsyymi hyödyntää kahdenarvoisia metalli-ioneja, luonnossa magnesiumia, joista kaksi on pysyvästi sitoutunut entsyymiin ja kaksi saapuu substraatin mukana. Reaktioon kuuluu nukleofiilisen happiatomin aktivointi ja tuotteen poistumiseen on useita mahdolisia reittejä. Tässä työssä olen määrittänyt muokkaamattoman hiivan pyrofosfataasin ja seitsemän variantin kiderakenteet magnesiumin kanssa. Nämä rakenteet selittävät mutaatioden vaikutukset katalyysiin ja mahdollistivat reaktion jokaisen välituotteen rakenteellisen kuvauksen. Vaikka tämä 'atomikoreografia' onkin tärkeä askel mekanismin ymmärtämisessä, ovat vetyatomit keskeisessä osassa mekanismissa. Yksikäsitteisin menetelmä vetyatomien paikkojen määrittämiseen on neutronikristallografia. Hiivan pyrofosfataasin neutronirakenteen määrittämiseksi tuotin perdeuteroitua entsyymiä ja kasvatin siitä suuria kiteitä. Koska kiteet eivät olleet stabiileja huoneenlämmössä, kehitetttin neutronimittauksia varten jäähdytyslaite. Tutkittasessa rakenteellisia muutoksia reaaliajassa aikaerotteisen kristallografian avulla tarvitaan fotolysoitava substraatin esiaste. Syntetisoin tällaisen ehdokasmolekyylin ja selvitin sen fotolyysikinetiikan, mutta valitettavasti sekä hiivan että Thermotoga maritiman pyrofosfataasit hydrolysoivat tämän molekyylin. Perheen II pyrofosfataasien mekanismi on hieman erilainen kuin ensimmäisellä perheellä, erityisesti metalliaktivaation osalta. Määritin Bacillus subtiliksen pyrofosfataasin rakenteen inhibiittori imidodifosfaatin kanssa sekä inaktiivisen H98Q variantin rakenteen pyrofosfaatin kanssa. Näistä rakenteista paljastui uudenlainen kolmen metalli-ionin muodostelma nukleofiilin aktivoimiseen.
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    ABSTRACT: Escherichia coli inorganic pyrophosphatase is a tight hexamer of identical subunits. Replacement of both His136 and His140 by Gln in the subunit interface results in an enzyme which is trimeric up to 26 mg/mL enzyme concentration in the presence of Mg2+, allowing direct measurements of Mg2+ binding to trimer by equilibrium dialysis. The results of such measurements, together with the results of activity measurements as a function of [Mg2+] and pH, indicate that Mg2+ binds more weakly to one of the three sites per monomer than it does to the equivalent site in the hexamer, suggesting this site to be located in the trimer:trimer interface. The otherwise unobtainable hexameric variant enzyme readily forms in the presence of magnesium phosphate, the product of the pyrophosphatase reaction, but rapidly dissociates on dilution into medium lacking magnesium phosphate or pyrophosphate. The kcat values are similar for the variant trimer and hexamer, but Km values are 3 orders of magnitude lower for the hexamer. Thus, while stabilizing hexamer, the two His residues, per se, are not absolutely required for active-site structure rearrangement upon hexamer formation. The reciprocal effect of hexamerization and product binding to the active site is explained by destabilization of alpha-helix A, contributing both to the active site and the subunit interface.
    Biochemistry 02/1998; 37(2):734-40. DOI:10.1021/bi9714823 · 3.01 Impact Factor
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