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Titolo:
DETERMINANTS OF COFACTOR SPECIFICITY IN ISOCITRATE DEHYDROGENASE - STRUCTURE OF AN ENGINEERED NADP(-]NAD(+) SPECIFICITY-REVERSAL MUTANT())
Autore:
HURLEY JH; CHEN RD; DEAN AM;
Indirizzi:
NIDDKD,MOLEC BIOL LAB,NATL INST HLTH BETHESDA MD 20892 CHICAGO MED SCH,DEPT BIOL CHEM N CHICAGO IL 60064
Titolo Testata:
Biochemistry
fascicolo: 18, volume: 35, anno: 1996,
pagine: 5670 - 5678
SICI:
0006-2960(1996)35:18<5670:DOCSII>2.0.ZU;2-U
Fonte:
ISI
Lingua:
ENG
Soggetto:
SACCHAROMYCES-CEREVISIAE; NUCLEOTIDE-SEQUENCE; 3-ISOPROPYLMALATE DEHYDROGENASE; THERMUS-THERMOPHILUS; COENZYME SPECIFICITY; GENE; PHOSPHORYLATION; ENZYME; MUTAGENESIS; DISRUPTION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
41
Recensione:
Indirizzi per estratti:
Citazione:
J.H. Hurley et al., "DETERMINANTS OF COFACTOR SPECIFICITY IN ISOCITRATE DEHYDROGENASE - STRUCTURE OF AN ENGINEERED NADP(-]NAD(+) SPECIFICITY-REVERSAL MUTANT())", Biochemistry, 35(18), 1996, pp. 5670-5678

Abstract

The 7-fold mutation /Lys344Asp/Tyr345Ile/Val351Ala/Tyr391Lys/Arg395Ser converts the cofactor specificity of Escherichia coli isocitrate dehydrogenase from a 7000-fold preference for NADP(+) to a 200-fold preference for NAD(+), with overall activity comparable to that of wild-type NAD(+)-dependent isocitrate dehydrogenases. The structure of the NAD(+)-dependent mutant has been determined and refined to a working R-factor of 0.186 at 1.9 Angstrom resolution. The structure shows that NADP(+) affinity is destroyed by removing favorable interactions between the 2'-phosphate and Tyr345, Tyr391, and Arg395 and by adding a repulsive interaction with Asp344. NAD(+) affinity is enhanced by adding hydrogen bonds between Asp344 and the free 2'-hydroxyl. The favorable Asp344-2'-OH interaction requires a change in the pucker of the ribose toC2' endo and a shift in the adenine ring, The ring shift is made possible by a series of changes in steric packing interactions. The linchpin for repacking in the adenosine binding site is residue 351, The side chain of this ''second layer'' residue dictates packing of the surrounding ''first layer'' residues which interact with the 2' moiety and,in turn, directly determine specificity.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 18/09/20 alle ore 20:06:47