117e
From Proteopedia
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==Overview== | ==Overview== | ||
| - | We have solved the structure of two active-site variants of soluble, inorganic pyrophosphatases (PPase), R78K and D117K, at resolutions of 1.85, and 2.15 A and R-factors of 19.5% and 18.3%, respectively.In the R78K, variant structure, the high-affinity phosphate group (P1) is missing, consistent with the wild-type structure showing a bidentate interaction, between P1 and Arg78, and solution data showing a decrease in P1 affinity, in the variant. The structure explains why the mutation affects P1 and, pyrophosphate binding much more than would be expected by the loss of one, hydrogen bond: Lys78 forms an ion-pair with Asp71, precluding an, interaction with P1. The R78K variant also provides the first direct, evidence that the low-affinity phosphate group (P2) can adopt the, structure that .. | + | We have solved the structure of two active-site variants of soluble, inorganic pyrophosphatases (PPase), R78K and D117K, at resolutions of 1.85, and 2.15 A and R-factors of 19.5% and 18.3%, respectively.In the R78K, variant structure, the high-affinity phosphate group (P1) is missing, consistent with the wild-type structure showing a bidentate interaction, between P1 and Arg78, and solution data showing a decrease in P1 affinity, in the variant. The structure explains why the mutation affects P1 and, pyrophosphate binding much more than would be expected by the loss of one, hydrogen bond: Lys78 forms an ion-pair with Asp71, precluding an, interaction with P1. The R78K variant also provides the first direct, evidence that the low-affinity phosphate group (P2) can adopt the, structure that we believe is the immediate product of hydrolysis, with one, of the P2 oxygen atoms co-ordinated to both activating metal ions (M1 and, M2). If so, the water molecule (Wat1) between M1 and M2 in wild-type PPase, is, indeed, the attacking nucleophile.The D117E variant structure likewise, supports our model of catalysis, as the Glu117 variant carboxylate group, is positioned where Wat1 is in the wild-type: the potent Wat1 nucleophile, is replaced by a carboxylate co-ordinated to two metal ions. Alternative, confirmations of Glu117 may allow Wat1 to be present but at much reduced, occupancy, explaining why the pKa of the nucleophile increases by three pH, units, even though there is relatively little distortion of the active, site.These new structures, together with parallel functional studies, measuring catalytic efficiency and ligand (metal ion, PPi and Pi) binding, provide strong evidence against a proposed mechanism in which Wat1 is, considered unimportant for hydrolysis. They thus support the notion that, PPase shares mechanistic similarity with the "two-metal ion" mechanism of, polymerases. |
==About this Structure== | ==About this Structure== | ||
| - | 117E is a | + | 117E is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae] with MN and PO4 as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Inorganic_diphosphatase Inorganic diphosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.6.1.1 3.6.1.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=117E OCA]. |
==Reference== | ==Reference== | ||
The R78K and D117E active-site variants of Saccharomyces cerevisiae soluble inorganic pyrophosphatase: structural studies and mechanistic implications., Tuominen V, Heikinheimo P, Kajander T, Torkkel T, Hyytia T, Kapyla J, Lahti R, Cooperman BS, Goldman A, J Mol Biol. 1998 Dec 18;284(5):1565-80. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=9878371 9878371] | The R78K and D117E active-site variants of Saccharomyces cerevisiae soluble inorganic pyrophosphatase: structural studies and mechanistic implications., Tuominen V, Heikinheimo P, Kajander T, Torkkel T, Hyytia T, Kapyla J, Lahti R, Cooperman BS, Goldman A, J Mol Biol. 1998 Dec 18;284(5):1565-80. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=9878371 9878371] | ||
| + | [[Category: Inorganic diphosphatase]] | ||
[[Category: Saccharomyces cerevisiae]] | [[Category: Saccharomyces cerevisiae]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
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[[Category: mutan structures]] | [[Category: mutan structures]] | ||
| - | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 12 15:50:43 2007'' |
Revision as of 13:44, 12 November 2007
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THE R78K AND D117E ACTIVE SITE VARIANTS OF SACCHAROMYCES CEREVISIAE SOLUBLE INORGANIC PYROPHOSPHATASE: STRUCTURAL STUDIES AND MECHANISTIC IMPLICATIONS
Overview
We have solved the structure of two active-site variants of soluble, inorganic pyrophosphatases (PPase), R78K and D117K, at resolutions of 1.85, and 2.15 A and R-factors of 19.5% and 18.3%, respectively.In the R78K, variant structure, the high-affinity phosphate group (P1) is missing, consistent with the wild-type structure showing a bidentate interaction, between P1 and Arg78, and solution data showing a decrease in P1 affinity, in the variant. The structure explains why the mutation affects P1 and, pyrophosphate binding much more than would be expected by the loss of one, hydrogen bond: Lys78 forms an ion-pair with Asp71, precluding an, interaction with P1. The R78K variant also provides the first direct, evidence that the low-affinity phosphate group (P2) can adopt the, structure that we believe is the immediate product of hydrolysis, with one, of the P2 oxygen atoms co-ordinated to both activating metal ions (M1 and, M2). If so, the water molecule (Wat1) between M1 and M2 in wild-type PPase, is, indeed, the attacking nucleophile.The D117E variant structure likewise, supports our model of catalysis, as the Glu117 variant carboxylate group, is positioned where Wat1 is in the wild-type: the potent Wat1 nucleophile, is replaced by a carboxylate co-ordinated to two metal ions. Alternative, confirmations of Glu117 may allow Wat1 to be present but at much reduced, occupancy, explaining why the pKa of the nucleophile increases by three pH, units, even though there is relatively little distortion of the active, site.These new structures, together with parallel functional studies, measuring catalytic efficiency and ligand (metal ion, PPi and Pi) binding, provide strong evidence against a proposed mechanism in which Wat1 is, considered unimportant for hydrolysis. They thus support the notion that, PPase shares mechanistic similarity with the "two-metal ion" mechanism of, polymerases.
About this Structure
117E is a Single protein structure of sequence from Saccharomyces cerevisiae with MN and PO4 as ligands. Active as Inorganic diphosphatase, with EC number 3.6.1.1 Full crystallographic information is available from OCA.
Reference
The R78K and D117E active-site variants of Saccharomyces cerevisiae soluble inorganic pyrophosphatase: structural studies and mechanistic implications., Tuominen V, Heikinheimo P, Kajander T, Torkkel T, Hyytia T, Kapyla J, Lahti R, Cooperman BS, Goldman A, J Mol Biol. 1998 Dec 18;284(5):1565-80. PMID:9878371
Page seeded by OCA on Mon Nov 12 15:50:43 2007
Categories: Inorganic diphosphatase | Saccharomyces cerevisiae | Single protein | Cooperman, B.S. | Goldman, A. | Heikinheimo, P. | Hyytia, T. | Kajander, T. | Kapyla, J. | Lahti, R. | Torkkel, T. | Tuominen, V. | MN | PO4 | 2-metal ion mechanism | Enzyme mechanism | Hydrolase | Iorganic pyrophosphatase | Mutan structures
