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Titolo:
THE ROLE OF GLUTAMATE-87 IN THE KINETIC MECHANISM OF THERMUS-THERMOPHILUS ISOPROPYLMALATE DEHYDROGENASE
Autore:
DEAN AM; DVORAK L;
Indirizzi:
CHICAGO MED SCH,DEPT BIOL CHEM,3333 GREEN BAY RD N CHICAGO IL 60064
Titolo Testata:
Protein science
fascicolo: 10, volume: 4, anno: 1995,
pagine: 2156 - 2167
SICI:
0961-8368(1995)4:10<2156:TROGIT>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEOTIDE-ISOCITRATE DEHYDROGENASE; 3-ISOPROPYLMALATE DEHYDROGENASE; EXTREME THERMOPHILE; ACTIVE-SITE; PHOSPHORYLATION; ENZYME; INHIBITION; SUBSTRATE;
Keywords:
IMDH; KINETIC MECHANISM; STABILIZATION OF MICHAELIS COMPLEX;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
31
Recensione:
Indirizzi per estratti:
Citazione:
A.M. Dean e L. Dvorak, "THE ROLE OF GLUTAMATE-87 IN THE KINETIC MECHANISM OF THERMUS-THERMOPHILUS ISOPROPYLMALATE DEHYDROGENASE", Protein science, 4(10), 1995, pp. 2156-2167

Abstract

The kinetic mechanism of the oxidative decarboxylation of 2R,3S-isopropylmalate by the NAD-dependent isopropylmalate dehydrogenase of Thermus thermaphilus was investigated. Initial rate results typical of random or steady-state ordered sequential mechanisms are obtained for boththe wild-type and two mutant enzymes (E87G and E87Q) regardless of whether natural or alternative substrates (2R-malate, 2R,3S-tartrate and/or NADP) are utilized. Initial rate data fail to converge on a rapid equilibrium-ordered pattern despite marked reductions in specificity (k(cat)/K-m) caused by the mutations and alternative substrates. Although the inhibition studies alone might suggest an ordered kinetic mechanism with cofactor binding first, a detailed analysis reveals that theexpected noncompetitive patterns appear uncompetitive because the dissociation constants from the ternary complexes are far smaller than those from the binary complexes. Equilibrium fluorescence studies both confirm the random binding of substrates and the kinetic estimates of the dissociation constants of the substrates from the binary complexes. The latter are not disturbed markedly by the mutations at site 87. Mutations at site 87 do not affect the dissociation constants from the binary complexes, but do greatly increase the Michaelis constants, indicating that E87 helps stabilize the Michaelis complex of the wild-typeenzyme. The available structural data, the patterns of the kinetics results, and the structure of a pseudo-Michaelis complex of the homologous isocitrate dehydrogenase of Escherichia col i suggest that E87 interacts with the nicotinamide ring.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 24/10/20 alle ore 11:49:12