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Titolo:
A HIGHLY-ACTIVE DECARBOXYLATING DEHYDROGENASE WITH RATIONALLY INVERTED COENZYME SPECIFICITY
Autore:
CHEN RD; GREER A; DEAN AM;
Indirizzi:
CHICAGO MED SCH,DEPT BIOL CHEM,3333 GREEN BAY RD N CHICAGO IL 60064
Titolo Testata:
Proceedings of the National Academy of Sciences of the United Statesof America
fascicolo: 25, volume: 92, anno: 1995,
pagine: 11666 - 11670
SICI:
0027-8424(1995)92:25<11666:AHDDWR>2.0.ZU;2-L
Fonte:
ISI
Lingua:
ENG
Soggetto:
ISOCITRATE DEHYDROGENASE; MALATE-DEHYDROGENASE; ESCHERICHIA-COLI; SITE; PHOSPHORYLATION; PURIFICATION; MUTAGENESIS; MECHANISM; PHOSPHATE; REDESIGN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
24
Recensione:
Indirizzi per estratti:
Citazione:
R.D. Chen et al., "A HIGHLY-ACTIVE DECARBOXYLATING DEHYDROGENASE WITH RATIONALLY INVERTED COENZYME SPECIFICITY", Proceedings of the National Academy of Sciences of the United Statesof America, 92(25), 1995, pp. 11666-11670

Abstract

The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of k(cat)/K-m). Guidedby x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosinebinding pocket that systematically shift coenzyme preference toward NAD, The engineered enzyme displays an 850-fold preference for NAD overNADP, which exceeds the 140-fold preference displayed by a homologousNAD-dependent enzyme, Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences-strict adherenceto homology as a criterion for replacing these amino acids impairs function, Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologousNAD-dependent enzymes.

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Documento generato il 26/09/20 alle ore 13:41:01