Effects of self-association of ornithine aminotransferase on its physicochemical characteristics.
Previous work in this laboratory [e.g., Peraino, C., Bunville, L. G., & Tahmisian, T. N. (1969) J. Biol. Chem. 244, 2241--2249, and Morris, J. E., Peraino, C., & Strayer, D. (1974) Proc. Soc. Exp. Biol. Med. 147, 707--709] has shown that the molecular weight of ornithine aminotransferase (OAT) is concentration dependent. In the present study this property of OAT was further characterized by using sedimentation equilibrium centrifugation to determine the molecular weight of OAT in a range of enzyme concentrations. It was shown that OAT aggregates in a two-stage process as its concentration increases. The first stage involves the association of enzymatically active monomers into trimers, with association of the trimers into higher order aggregates occurring in the second stage. Decreasing the pH or raising the ionic strength enhances aggregation while raising the pH inhibits aggregation; however, the two-stage nature of the aggregation process was not affected by changes in pH and ionic strength. Kinetic analyses of purified enzyme showed that aggregation results in an increase in the kM for both substrates with the Vmax remaining constant, indicating that aggregation of monomers sterically hinders substrate binding. Increased Km values were also obtained for OAT sequestered in mitochondria from rats fed a high-protein diet to increase mitochondrial OAT levels. The higher Km values suggest that the elevation of OAT in vivo is accompanied by aggregation of the enzyme within the mitochondrion. We propose that the aggregation-dependent increase of Km in vivo has adaptive value in that it spares ornithine for use in the urea cycle.
D000818:Animals; D007656:Ketoglutaric Acids; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008433:Mathematics; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D009952:Ornithine; D009953:Ornithine-Oxo-Acid Transaminase / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding; D051381:Rats; D000637:Transaminases / Q000378:metabolism*
D007656:Ketoglutaric Acids; D046911:Macromolecular Substances; D009952:Ornithine; D000637:Transaminases; D009953:Ornithine-Oxo-Acid Transaminase