Ornithine transcarbamylase from Salmonella typhimurium: purification, subunit composition, kinetic analysis, and immunological cross-reactivity

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Abstract
Ornithine transcarbamylase (OTCase) [EC 2.1.3.3] was purified to homogeneity from a derepressed strain of S. typhimurium. The optimal pH for enzyme activity is 8.0. The MW of the enzyme was 116,000 based on measurements of the sedimentation coefficient by sucrose gradient ultracentrifugation and the Stokes radius by gel filtration. Polyacrylamide gel electrophoresis of cross-linked OTCase in the presence of sodium dodecyl sulfate showed that the enzyme is composed of 3 identical subunits. The MW of the monomer was 39,000. Steady-state kinetics indicate that the reaction mechanism is sequential. The limiting Km for carbamylphosphate and ornithine were 0.06 and 0.2 mM, respectively. The Kd for carbamylphosphate was 0.02 mM. Product and dead-end inhibition patterns are consistent with an ordered Bi Bi mechanism, in which carbamylphosphate is the first substrate added and phosphate is the last product released. OTCase activity was inhibited by arginine, but relatively high concentrations were required for significant inhibition. The inhibition by arginine might be physiologically significant in the regulation of carbamylphosphate utilization; a single carbamylphosphate synthetase is responsible for the synthesis of carbamylphosphate for arginine and pyrimidines in S. typhimurium, and the inhibition by arginine might serve to divert carbamylphosphate to the synthesis of pyrimidines when arginine is present at high concentrations. The cross-reaction of OTCases from different microorganisms with purified [rabbit] antibodies raised against the homogeneous OTCase from S. typhimurium was investigated. The results of immunotitration and immunodiffusion experiments revealed a high degree of identity between the enzymes from S. typhimurium and Escherichia coli B and W. In these 3 cases, a single gene (argI)encodes OTCase. Wild-type E. coli K-12 and strain 3000 X 111, which carry 2 OTCase genes (argI, argF), also revealed similar cross-reactivity, indicating that argF is the product of a relatively recent [evolutionary] duplication. The activity of OTCase from Bacillus subtilis was partially inhibited by antibodies against the enzyme from S. typhimurium, indicating unusual conservation of primary structure among widely different taxonomic groups. OTCase from Saccharomyces cerevisiae, whose MW and primary structure are similar to those of the enzyme from S. typhimurium, was without detectable cross-reactivity.