Methionine-321 in the C-terminal alpha-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity.

Details

Serval ID
serval:BIB_ECEA77FBC027
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Methionine-321 in the C-terminal alpha-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity.
Journal
FEMS Microbiology Letters
Author(s)
Nguyen V.T., Tricot C., Stalon V., Dideberg O., Villeret V., Haas D.
ISSN
0378-1097 (Print)
ISSN-L
0378-1097
Publication state
Published
Issued date
1994
Volume
124
Number
3
Pages
411-417
Language
english
Abstract
Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]0.5) and high cooperativity toward this substrate. We have isolated mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa. Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphosphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.
Keywords
Allosteric Regulation, Animals, Enzyme Activation/genetics, Methionine/chemistry, Mutation, Ornithine Carbamoyltransferase/antagonists & inhibitors, Ornithine Carbamoyltransferase/genetics, Plasmids, Protein Structure, Secondary, Pseudomonas aeruginosa/enzymology, Pseudomonas aeruginosa/genetics
Pubmed
Web of science
Create date
25/01/2008 18:01
Last modification date
20/08/2019 17:14
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