Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme.

Détails

ID Serval
serval:BIB_4556DEAED688
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme.
Périodique
Journal of Biological Chemistry
Auteur(s)
Baur H., Tricot C., Stalon V., Haas D.
ISSN
0021-9258 (Print)
ISSN-L
0021-9258
Statut éditorial
Publié
Date de publication
1990
Peer-reviewed
Oui
Volume
265
Numéro
25
Pages
14728-14731
Langue
anglais
Résumé
Pseudomonas aeruginosa has an anabolic and a catabolic ornithine carbamoyltransferase (OTCase). In vitro, these homologous enzymes catalyze the same reaction (ornithine + carbamoyl phosphate (CP) in equilibrium citrulline + Pi), yet in vivo they function unidirectionally owing to specific kinetic properties. The catabolic OTC-ase cannot promote the anabolic reaction (citrulline formation) in vivo because of a sigmoidal CP saturation curve and a high CP concentration for half-maximal velocity. The structural basis for this kinetic specialization was examined. The catabolic OTCase lost most of its homotropic cooperativity and gained anabolic activity when an amino acid residue near the CP binding site, Glu-106, was replaced by alanine or glycine. In the anabolic OTCase of Escherichia coli the glutamine residue corresponding to Glu-106 was exchanged for glutamate; however, in this case no CP cooperativity was acquired. Thus, in catabolic OTCase, sequence features in addition to Glu-106 are important for sigmoidal CP saturation, and such a sequence was identified in the C-terminal part. By an in vivo gene fusion technique the 9 C-terminal amino acids of catabolic OTCase were replaced by the homologous 8 amino acids from anabolic OTCase of E. coli; the hybrid enzyme had a markedly reduced homotropic cooperativity. This gene fusion method should be generally useful for directed enzyme evolution.
Mots-clé
Amino Acid Sequence, Base Sequence, Escherichia coli/enzymology, Escherichia coli/genetics, Genotype, Molecular Sequence Data, Mutation, Ornithine Carbamoyltransferase/genetics, Ornithine Carbamoyltransferase/metabolism, Pseudomonas aeruginosa/enzymology, Pseudomonas aeruginosa/genetics, Restriction Mapping, Transduction, Genetic
Pubmed
Web of science
Création de la notice
25/01/2008 18:01
Dernière modification de la notice
03/03/2018 16:45
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