Zn2+ regulation of ornithine transcarbamoylase. I. Mechanism of action.
Ornithine transcarbamoylase catalyzes the formation of L-citrulline from carbamoyl phosphate and L-ornithine. The anabolic enzyme from Escherichia coli is composed of three identical subunits and resembles, in both primary and quaternary structures, the catalytic unit of aspartate transcarbamoylase. However, ornithine transcarbamoylase has no regulatory subunits. Although this enzyme does not bind its substrates co-operatively, fluorescence spectroscopic experiments show that zinc adds allosterically to the free enzyme. The metal binding process is dictated by deprotonation of an enzymic group with a pKa less than 7. The saturation binding curve of the metal ion is sigmoidal and yields a Hill coefficient of 1.6 at pH 8.5 and 25 degrees C. In the absence of substrates, zinc further promotes a slow enzyme isomerization, which occurs with a first-order rate constant of 7 min-1; thus, the metal is a slow, tight-binding inhibitor. The isomerized enzyme is inactive and contains three zinc ions. When the enzyme is first bound with carbamoyl phosphate, steady-state kinetic assays reveal that zinc again binds co-operatively to the binary enzyme complex, with a Hill coefficient of 1.5, but the metal ion now behaves simply as a classical, reversible inhibitor; it is competitive against L-ornithine, non-competitive against carbamoyl phosphate, and induces no enzyme isomerization. However, as a result of the competition between zinc and L-ornithine for the same site on the enzyme, the L-ornithine saturation curve becomes sigmoidal. Displacement of the allosteric zinc from the enzyme by L-ornithine is the cause of co-operative addition of this substrate. The combined results suggest that zinc regulates ornithine transcarbamoylase via two routes: (1) as an allosteric cofactor of the substrate-bound enzyme in mediating site-site interactions; and (2) as a slow, tight-binding inhibitor of the free enzyme in inducing inactivation. The concentration of zinc that is effective for action is in the micromolar range. The finding that E. coli ornithine transcarbamoylase can be induced to express co-operativity in binding its substrates has recently been confirmed by site-directed mutagenesis experiments. When the active site residue Arg106 is altered to a glycine, the resultant mutant enzyme exhibits both homotropic and heterotropic interactions towards its substrates. In view of the quaternary structure of holoenzyme aspartate transcarbamoylase, the "silent" co-operativity of ornithine transcarbamoylase is of particular interest in the study of evolution of complex, regulatory proteins.
Lee
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Department of Chemistry, Metcalf Center for Science & Engineering, Boston University, MA 02215.
Shen
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Miller
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Kuo
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LC
D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008433:Mathematics; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000494:pharmacology*
D008024:Ligands; D046911:Macromolecular Substances; D004492:Edetic Acid; D009954:Ornithine Carbamoyltransferase; D015032:Zinc