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

Abstract

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.

Quaternary structure of ornithine aminotransferase in solution and preliminary crystallographic data

“…Most of the M I values for the oligomeric protein have varied in the range 116000 -202000 [6][7][8]15, 201 with many of the estimates being at the upper end of this range, thus leading to the conclusion that the enzyme is a tetramer [6, 7, 151. Our experimental findings strongly favour the existence of the enzyme as a hexamer in solution: (a) the M , of OAT in solution is rather constant (256000) over a broad range of protein concentration in the presence of sodium chloride; (b) cross-linking experiments never yielded aggregates containing more than six units even at high protein concentrations; and (c) crystallographic threefold symmetry is compatible with a molecular symmetry of a hexamer but not of a tetramer. These results support the data of Boernke et al [5] but only in part since we have failed to find any evidence that continuous aggregation of trimers to oligomers larger than hexamers exists. Their model implies a mixed population of trimers, hexamers, nanomers, etc.…”

“…These results are in agreement with the data of Boernke et al [5]. The higher aggregational state of OAT in the presence of NaCl reported by earlier workers [5] and confirmed by our experiments (Fig.…”

“…The equilibration by vapour diffusion concentration over 0.1 -3.0 mg/ml. The latter behaviour was confirmed by Boernke et al [5] who suggested that it was due to a continuous process of aggregation through the addition of further trimeric units to give hexamers, nanomers and higher oligomers. Their data, however, do not show the presence of any species with a molecular mass significantly above that expected for a hexamer.…”

“…The pH interval optimal for crystallization was 7.2-8. 5. Outside of this pH range the crystals did not grow.…”

High‐resolution Electron Microscopic Studies on the Quaternary Structure of Ornithine Aminotransferase from Pig Kidney

“…Peraino et al (1969) and Morris et al (1974) stated that the molecular mass of rat liver OAT depends on the protein concentration. Boernke et al (1981) assumed a two-step process of aggregation responsible for the existence of 130-140-kDa trimers at the first stage, running finally to the hexamer of 280 kDa in the second stage of aggregation. Boernke et al (1982) believed that the aggregation attributes to the positive charges on the monomer surface and loss of these positive surface charges prevents extensive self-aggregation.…”