Effects of self-association of ornithine aminotransferase on its physicochemical characteristics

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Abstract
Previous work showed that the MW 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 MW of OAT in a range of enzyme concentrations. OAT aggregates in a 2-stage process as its concentration increases. The 1st stage involves the association of enzymatically active monomers into trimers, with association of the trimers into higher order aggregates occurring in the 2nd stage. Decreasing the pH or raising the ionic strength enhances aggregation while raising the pH inhibits aggregation; the 2-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 obtained for OAT sequestered in liver mitochondria from rats fed a high-protein diet to increase mitochondrial OAT levels. The higher Km values suggest that elevation of OAT in vivo is accompanied by aggregation of the enzyme within the mitochondrion. The aggregation-dependent increase of Km in vivo may have adaptive value in that it spares ornithine for use in the urea cycle.