1grp | pdb_00001grp

REGULATORY AND CATALYTIC MECHANISMS IN ESCHERICHIA COLI ISOCITRATE DEHYDROGENASE: MULTIPLE ROLES FOR N115

spinqualitylabelsall models 1grp, resolution 2.50Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 1grp is a 1 chain structure with sequence from "bacillus_coli"_migula_1895 "bacillus coli" migula 1895. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: ICT, MG Activity: Isocitrate dehydrogenase (NADP(+)), with EC number 1.1.1.42 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed Inactivation of Escherichia coli isocitrate dehydrogenase upon phosphorylation at S113 depends upon the direct electrostatic repulsion of the negatively charged gamma-carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (kcat/K(i).isocitrat/ Km.NADP) by a factor of 10(7). Here, we demonstrate that the inactivating effects of the electrostatic repulsion are completely eliminated by a second-site mutation, and provide the structural basis for this striking example of intragenic suppression. N115 is adjacent to S113 on one face of the D-helix, interacts with isocitrate and NADP+, and has been postulated to serve in both substrate binding and in catalysis. The single N115L substitution reduces affinity for isocitrate by a factor of 50 and performance by a factor of 500. However, the N115L substitution completely suppresses the inactivating electrostatic effects of S113D or S113E: the performance of the double mutants is 10(5) higher than the S113D and S113E single mutants. These mutations have little effect on the kinetics of alternative substrates, which lack the charged gamma-carboxylate of isocitrate. Both glutamate and aspartate at site 113 remain fully ionized in the presence of leucine. In the crystal structure of the N115L mutant, the leucine adopts a different conformer from the wild-type asparagine. Repacking around the leucine forces the amino-terminus of the D-helix away from the rest of the active site. The hydrogen bond between E113 and N115 in the S113E single mutant is broken in the S113E/N115L mutant, allowing the glutamate side chain to move away from the gamma-carboxylate of isocitrate. These movements increase the distance between the carboxylates, diminish the electrostatic repulsion, and lead to the remarkably high activity of the S113E/N115L mutant. Second-site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase.,Chen R, Grobler JA, Hurley JH, Dean AM Protein Sci. 1996 Feb;5(2):287-95. PMID:8745407 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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