Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
Threonine 82 in the regulatory chain is important for nucleotide affinity and for the allosteric stabilization of Escherichia coli aspartate transcarbamoylase
Autore:
Williams, MK; Kantrowitz, ER;
Indirizzi:
Boston Coll, Dept Chem, Merkert Chem Ctr, Chestnut Hill, MA 02167 USA Boston Coll Chestnut Hill MA USA 02167 m Ctr, Chestnut Hill, MA 02167 USA
Titolo Testata:
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
fascicolo: 1, volume: 1429, anno: 1998,
pagine: 249 - 258
SICI:
0167-4838(199812)1429:1<249:T8ITRC>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
SITE-SPECIFIC MUTAGENESIS; RAY SOLUTION SCATTERING; CRYSTAL-STRUCTURES; EFFECTOR BINDING; SYNERGISTIC INHIBITION; PHENOTYPIC SELECTION; 2.8-A RESOLUTION; NEUTRAL PH; LIGATED-T; CTP;
Keywords:
site-specific mutagenesis; heterotropic interaction; allosteric enzyme;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
38
Recensione:
Indirizzi per estratti:
Indirizzo: Kantrowitz, ER Boston Coll, Dept Chem, Merkert Chem Ctr, Chestnut Hill, MA02167 USA Boston Coll Chestnut Hill MA USA 02167 Hill, MA 02167 USA
Citazione:
M.K. Williams e E.R. Kantrowitz, "Threonine 82 in the regulatory chain is important for nucleotide affinity and for the allosteric stabilization of Escherichia coli aspartate transcarbamoylase", BBA-PROT ST, 1429(1), 1998, pp. 249-258

Abstract

The three-dimensional structure of Escherichia call aspartate transcarbamoylase complexed with the allosteric effector CTP, shows an interaction between the hydroxyl of Thr-82 in the regulatory chain (Thr-82r) with the gamma-phosphate of CTP (R.P. Kosman, J.E. Gouaux, W.N. Lipscomb, Crystal structure of. CTP-ligated T state aspartate transcarbamoylase at 2.5 Angstrom resolution: implications for aspartate transcarbamoylase mutants and the mechanism of negative cooperativity, Proteins Struct. Funct. Genet. 15 (1993) 147-176). In order to determine whether the Thr-82r interaction with the gamma-phosphate of CTP is important for either binding of the nucleotide effecters or their function, site-specific mutagenesis was employed. The mutant enzyme in which Thr-82r was replaced by Ala had almost the identical maximal observed specific activity as the wildtype enzyme; however, the mutant enzyme had a significantly increased [Asp](0.5), the aspartate concentration atone-half the maximal observed specific activity, as well as slightly increased homotropic cooperativity. The mutant enzyme was also activated more byATP and inhibited less by CTP as compared to the wild-type enzyme. In addition, the nucleotide concentration required for one-half maximal effect wasincreased approx. 3-fold as compared to the corresponding values for the wild-type enzyme. The maximal inhibition of the mutant enzyme, in the presence of UTP and CTP was similar to that observed for the wild-type enzyme; however, higher concentrations of the nucleotides were required to achieve this level of inhibition. The reduced affinity of CTP, UTP and ATP induced bythe mutation indicates that the hydrogen bonding interaction between the gamma-phosphate of the nucleotide effector and the side-chain hydroxyl of Thr-82r is important for the binding of the nucleotide effecters to the allosteric site. Furthermore, this interaction is important for the discrimination between CTP and CDP. Finally, the greater homotropic cooperativity, greater [Asp](0.5), diminished CTP inhibition and greater ATP activation of themutant enzyme correlates with the X-ray structure of the mutant enzyme which shows that the unligated enzyme is in an 'extreme' T-state. These findings add support to the theory that the global stabilization of the enzyme iscritical for both the homotropic and heterotropic properties of aspartate transcarbamoylase. (C) 1998 Elsevier Science B.V. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 19/09/20 alle ore 12:53:26