Zn(II)-induced cooperativity of Escherichia coli ornithine transcarbamoylase

Abstract

Ornithine transcarbamoylase (OTCase) catalyses the transfer of the carbamoyl from carbamoyl phosphate to L-ornithine for the synthesis of L-citrulline in the urea cycle. The enzyme shares a common source of the carbamoyl group with aspartate transcarbamoylase (ATCase), which catalyzes a similar reaction in the pyrimidine biosynthesis pathway. Unlike the hexameric ATCase, anabolic OTCase is a trimeric molecule of 105,000 daltons and does not display sigmoidal substrate saturation curves. The steady state reaction of OTCase purified from E. coli K-12 (argR, argF) [1] exhibits Michaelis-Menten kinetics for both substrate. Carbamoyl phosphate is the first substrate bound. However, when the competitive inhibitor Zn(II) is present, this anabolic OTCase expresses positive cooperativity towards its second substrate. The extent of cooperativity is a function of Zn(II) concentration. Steady state kinetic data yield a limiting Hill coefficient of 2.7 for L-ornithine at 0.3 m M Zn(II). The allosteric effect of Zn(II) on the enzyme is reversible and is not altered by the level of carbamoyl phosphate. At fixed substrate concentrations, initial velocity data obtained at 0–0.3 m M Zn(II) indicate cooperative binding of the metal ion to OTCase; a Hill coefficient of 1.7 ± 0.1 is found. there results suggest that conformational changes are only induced in the subunits of the enzyme by the metal ligand. Consequently, the positive cooperativity observed for L-ornithine is a manifestation of the allosteric effect of Zn(II). This phenomenon arises as a result of displacement of the metal ion from the enzyme by the substrate. The interpretation is further supported by a theoretical treatment based on equations derived for the two-state MWC model under the condition of competition between a substrate (non-cooperative) and an inhibitor (cooperative). Our study reveals that substrate cooperativity mediated indirectly via a competitive metal inhibitor is a special case previously unrecognized in enzyme allosteric control. Because of the unique cooperative behavior of OTCase and its uncommon quaternary structure, it is of special interest to understand the mechanism through which subunit interactions are transmitted. For ATCase, the protein is composed of two trimeric catalytic subunits and three dimeric regulatory subunits; the catalytic trimer is devoid of cooperative action in the absence of the regulatory dimers [2]. In comparison and at least in vitro, Zn(II) appears to substitute functionally in lieu of a regulatory subunit in OTCase.