Ornithine carbamoyltransferase from Eschrichia coli W was purified to homogeneity. The enzyme has a molecular weight of 105000. It is composed of three apparently identical subunits with molecular weights of 35 000. The mechanism of the ornithine carbamoyltransferase enzyme system from E. coli W was investigated kinetically by using the approach of product inhibition and dead-end inhibition of both forward and reverse reactions. On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released. The same studies also indicate that the mechanism involves dead-end complexes.The reaction mechanism appears consistent with that proposed by Theorell and Chance. Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme. Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude.Ornithine carbamoyltransferase catalyzes the synthesis of citrulline from ornithine and carbamoylphosphate. This enzyme is present in all arginine prototrophic organisms and belongs to the arginine biosynthetic pathway. A few procaryotes, such as Pseudomonas and Lactohacteria, are able to degrade arginine by the so-called arginine dihydrolase pathway, where the deamination of the guanido group of arginine is followed by the phosphorolysis of citrulline. This latter step is the same in reverse as one of the reactions of the biosynthetic pathway. In Pseudomonas enzymes for both directions of the reaction were present simultaneously [I]. In vitro both Pseudornonas ornithine carbainoyltranferases catalyze citrulline synthesis, but only the catabolic transferase is able to catalyze the phosphorolysis of citrulline [2]. However a mutation, which affects the anabolic transferase, leads to auxotrophy for arginine in spite of the presence of the catabolic enzyme [2]. Therefore, although the carbamoylation of ornithine is thermodynamically the most favoured direction of the reaction, the catabolic enzyme only catalyzes the unfavourable direction. Consequently, both transferases Ennzymes. Ornilhine cdrbdtnoyltraiisferase (EC 2.1.3.3); carbamate kinase (EC 2.7.2.2). appear extremely specialized, because in vivo each enzyme catalyzes exclusively the reaction which corresponds to its function. The apparent irreversibility of the catabolic enzyme has been explained previously [l]. That of the anabolic enzyme is the subject of an article in preparation. It was shown earlier, however, that if the allosteric properties of the catabolic enzyme (which cause its irreversibility in vivo) are partiall) destroyed, the enzyme can be used for anabolic purposes [4,5].Except for the anabolic transferase of Pseudomonas, the anabolic enzymes of procaryotes and eucaryotes appear to ope...

The Pyrococcus furiosus (PF) ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heatstable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100°C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 Å and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis-Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.