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Titolo:
2ND-SITE SUPPRESSION OF REGULATORY PHOSPHORYLATION IN ESCHERICHIA-COLI ISOCITRATE DEHYDROGENASE
Autore:
CHEN RD; GROBLER JA; HURLEY JH; DEAN AM;
Indirizzi:
CHICAGO MED SCH,DEPT BIOL CHEM,3333 GREEN BAY RD N CHICAGO IL 60064 CHICAGO MED SCH,DEPT BIOL CHEM N CHICAGO IL 60064 NIDDKD,NIH,MOLEC BIOL LAB BETHESDA MD 20892
Titolo Testata:
Protein science
fascicolo: 2, volume: 5, anno: 1996,
pagine: 287 - 295
SICI:
0961-8368(1996)5:2<287:2SORPI>2.0.ZU;2-7
Fonte:
ISI
Lingua:
ENG
Soggetto:
ACTIVE-SITE; RANDOM MUTAGENESIS; ENZYME; RESOLUTION; MECHANISM; PHOSPHATE; PROGRAM; MUTANT; CHAIN;
Keywords:
INTRAGENIC SUPPRESSION; ISOCITRATE DEHYDROGENASE; REGULATORY PHOSPHORYLATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
36
Recensione:
Indirizzi per estratti:
Citazione:
R.D. Chen et al., "2ND-SITE SUPPRESSION OF REGULATORY PHOSPHORYLATION IN ESCHERICHIA-COLI ISOCITRATE DEHYDROGENASE", Protein science, 5(2), 1996, pp. 287-295

Abstract

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 withaspartate or glutamate, which reduce performance (k(cat)/Ki . 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 bya factor of 50 and performance by a factor of 500. However, the N115Lsubstitution 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 18/09/20 alle ore 18:50:40