Anabolic Ornithine Carbamoyltransferase of Escherichia coli and Catabolic Ornithine Carbamoyltransferase of Pseudomonas putida. Steady-State Kinetic Analysis

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Abstract
The anabolic and catabolic ornithine carbamoyltransferases of P. putida display a unidirectional catalytic specialization: in citrulline synthesis for the anabolic enzyme, in citrulline phosphorolysis for the catabolic one. The irreversibility of the anabolic enzyme in vitro was previously explained by its kinetic properties, but the irreversibility of the catabolic transferase in vivo was due to its allosteric behavior. A steady-state kinetic analysis was carried out on the catabolic ornithine carbamoyltransferase at pH 6.8 in the presence of the allosteric activator, phosphate. The kinetic mechanism of E. coli ornithine carbamoyltransferase serving as a reference was also determined. For the E. coli enzyme in the reverse direction, the initial velocity patterns converging on the abscissa were obtained with either citrulline or arsenate as variable substrate. The inhibition by the product ornithine was linear competitive with respect to citrulline and linear non-competitive with respect to arsenate. In the forward direction phosphate and its analogs induce an inhibition by ornithine which is partial and competitive with respect to carbamoylphosphate. Together with the results of thermoinactivation studies in the presence of each reactant, this observation suggests a random kinetic mechanism, but with most of the reaction flux following the path where carbamoylphosphate adds before ornithine, when substrates are present at Km levels. The allosteric catabolic ornithine carbamoyltransferase of Pseudomonas displays qualitatively the same pattern as the E. coli enzyme.