117e: Difference between revisions

Revision as of 12:44, 28 October 2007

THE R78K AND D117E ACTIVE SITE VARIANTS OF SACCHAROMYCES CEREVISIAE SOLUBLE INORGANIC PYROPHOSPHATASE: STRUCTURAL STUDIES AND MECHANISTIC IMPLICATIONS

OverviewOverview

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 ... [(full description)]

About this StructureAbout this Structure

117E is a [Protein single] structure of sequence from [SACCHAROMYCES CEREVISIAE] with MN and PO4 as [ligands].

ReferenceReference

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

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 ==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.
+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 ... [[http://ispc.weizmann.ac.il/pmbin/getpm?9878371 (full description)]]
 ==About this Structure==
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 [[Category: mutan structures]]
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