Effects of Anions on the Substrate Affinities of the Pyridoxal and Pyridoxamine Forms of Mitochondrial and Supernatant Aspartate Transaminases

The effects of anions on substrate binding to the mitochondrial and supernatant isozymes of aspartate transaminase (EC 2.6.1.1) have been studied by direct spectrophotometric titrations of the active site-bound pyridoxal phosphate. The binding affinity of both isozymes for amino and dicarboxylic acids is dependent on the type and concentration of buffer anions. At high physiological pH, the substrate dissociation constants are proportional to the anion concentration. At low pH, substrate binding to the enzyme involves the displacement of more than one anion. The anions act as competitive inhibitors of the substrates and are thought to occupy the positively charged sites at the isozymes' active center where the substrate(s) carboxyl group(s) bind. Of all the anions studied, the order of inhibition was benzoate g chloride g acetate g phosphate g cacodylate. The comparison of the dissociation constants of the enzyme-substrate complexes for the mitochondrial and supernatant isozymes confirms that, although there is competition between the anions and the substrates or dicarboxylic acid inhibitors for the active center of the enzyme, the anion independent dissociation constants for substrates and inhibitors are intrinsically different in each isozyme. The various buffer anions also affect the pK of the active site-bound pyridoxal phosphate chromophore in different ways. Instead of the pK values of 6.3 previously reported for the mitochondrial and supernatant chromophore in chloride or phosphate buffers (high affinity anions), a pK of 5.4 can be measured for both transaminases with cacodylate buffers (low affinity anions). Thus, the binding of anions results in a pK shift of the chromophore to higher pH. This shift seems to be more pronounced the higher the affinity of the enzyme for the anion. Anions also act as competitive inhibitors for the second half of the transamination, the conversion of the pyridoxamine enzyme to the pyridoxal enzyme. The anion-binding affinities of the pyridoxamine enzyme are higher than those for the pyridoxal enzyme in both supernatant and mitochondrial transminases. Thus, a given anion concentration will affect unequally the apparent affinity of the pyridoxal form for amino acid and of the pyridoxamine form for keto acid. Since in general, the mitochondrial enzyme shows a higher anion affinity than the supernatant isozyme, whether in the pyridoxal or pyridoxamine form, the anion competition with substrates at any given concentration will be greater for the mitochondrial isozyme. The mechanistic schemes of either isozyme of aspartate transaminase must, therefore, include anion effects. Besides the obligatory enzyme substrate intermediates, there must be complexes such as pyridoxal enzyme-anion and pyridoxamine enzyme-anions in equilibrium mixtures of aspartate transaminase, substrate(s), and buffers.