Steady-state kinetics and analysis of pH dependence on wild-type and a modified allosteric Pseudomonas aeruginosa ornithine carbamoyltransferase containing the replacement of glutamate 105 by alanine.
The substitution of alanine for glutamate at position 105 (E105A) of the allosteric ornithine carbamoyltransferase (OTCase) of Pseudomonas aeruginosa abolishes the carbamoylphosphate (CP) cooperativity observed in the wild-type enzyme. A kinetic analysis of [E105A]OTCase was performed in order to determine the mechanism of the reaction. The results of initial velocity and inhibition studies are consistent with an ordered mechanism with CP as the first substrate to add to the enzyme. In addition, similar studies have been made using the wild-type enzyme in the presence of the activator, phosphate. The results are similar to those obtained with [E105A]OTCase indicating that the residue E105 is critical for the allosteric transition of the wild-type enzyme. The activities of the wild-type allosteric OTCase and of [E105A]OTCase have been studied in the pH range 5.8-8.2 in the absence and in the presence of positive and negative effectors. The sigmoid saturation of OTCases by CP has been analyzed according to the Hill equation. At low pH values, CP cooperativity is low in the wild-type enzyme but cooperativity and [S]CP0.5 values increase markedly with pH. For [E105A]OTCase, the saturation by CP is hyperbolic at all pH values; in this modified enzyme, the presence of spermidine, an allosteric inhibitor of the wild-type enzyme, results in an inhibition which induces CP cooperativity. Thus, the ionization of the residue E105 apparently results in a conformational change in the wild-type enzyme which modifies the catalytic site. Since the [E105A] enzyme retains the heterotropic effects of the wild-type enzyme, other structural features are required for the allosteric transition in the wild-type catabolic OTCase.
Tricot
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Laboratoire de Microbiologie, Faculté des Sciences, Université Libre de Bruxelles, Belgique.
Nguyen
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VT
Stalon
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D000409:Alanine / Q000737:chemistry*; D000494:Allosteric Regulation; D005971:Glutamates / Q000737:chemistry*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity
D005971:Glutamates; D018698:Glutamic Acid; D009954:Ornithine Carbamoyltransferase; D000409:Alanine