Functional properties of ornithine-ketoacid aminotransferase from rat liver

Abstract

Functional properties of rat liver ornithine ketoacid aminotransferase ( l-ornithine:2-oxoacid aminotransferase, EC 2.6.1.13) were examined by two procedures: (A) The overall enzyme reaction was separated into its component half-reactions and the capacity of the enzyme to react with each of its substrates individually was investigated under different conditions. (B) The enzyme was exposed to pyridoxal phosphate, and resultant effects on enzyme activity and pyridoxal phosphate binding were analyzed. The following properties of the enzyme were disclosed by these studies. 1. 1. The reaction of the enzyme with ornithine alone is maximal between pH 8 and 10 whereas its reaction with α-ketoglutarate is maximal between pH 6 and 8. The pH optimum for the overall reaction (pH 8.15) coincides with the point of intersection of the curves for the half reactions. 2. 2. The pyridoxamine form of the enzyme, produced by the reaction of the pyridoxal enzyme with ornithine, reverts to the pyridoxal form in the absence of the second substrate. The rate of this reversion is lower in Tris than in phosphate buffer, although the catalytic activity of the enzyme is identical in both buffers. The lability of pyridoxamine at the active site is a consequence of its attachment to the apoenzyme, since free pyridoxamine phosphate does not release its amino group under the same conditions. It is suggested that this destabilizing effect of the apoenzyme is important to its role in the catalysis of the amino-transfer reaction. 3. 3. Exposure of the enzyme to excess pyridoxal phosphate is accompanied by the partial inactivation of the enzyme. This partial inactivation coincides with the binding of a small amount of pyridoxal phosphate to the enzyme. The further binding of pyridoxal phosphate produces no additional inactivation. Since pyridoxal phosphate reacts specifically with lysine residues in proteins the results suggest that certain of the lysine residues are involved in maintaining the enzyme at full catalytic capacity while others are functionally inert.