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Distinctions in the equilibrium kinetic constants of the mitochondrial and supernatant isozymes of aspartate transaminase.

C. M. Michuda,M. Martinez-Carrión

Published 1969 in Journal of Biological Chemistry

ABSTRACT

Abstract A detailed comparison of the interactions of substrates with the mitochondrial and supernatant isozymes of glutamateaspartate transaminase (EC 2.6.1.1) has been made by direct spectrophotometric titrations of the natural reporter, bound pyridoxal phosphate, at the active site. Spectroscopic methods are presented for the analysis of the dissociation constants of the aspartate-oxalacetate and glutamate-α-ketoglutarate equilibria of mitochondrial glutamate-aspartate transaminase. Mathematical treatment of the spectrophotometric data is based on the assumption that the mechanism involves only binary complexes. The agreement and linearity of the graphical results of these methods rule out site-site interactions or the formation of ternary complexes, such as enzyme-amino acid-keto acid. The pyridoxal form of the mitochondrial isozyme has a higher affinity for aspartate (K1 = 0.47 mm) than for glutamate (K1 = 12.4 mm). It binds oxalacetate (K3 = 46 mm) and α-ketoglutarate (K3 = 620 mm) as inactive abortive complexes. In the supernatant enzyme these values are aspartate, K1 = 4 mm; glutamate, K1 = 14 mm; oxalacetate, K3 = 100 mm; and α-ketoglutarate, K3 = 50 mm. The pyridoxamine form of both isozymes binds oxalacetate (K2 = 14 µm, mitochondrial; K2 = 20 µm, supernatant) better than α-ketoglutarate (K2 = 0.7 mm, mitochondrial; K2 = 0.4 mm, supernatant), but it has a low affinity for glutamate and aspartate. The substrate analogue, erythro-β-hydroxyaspartate, forms a complex(es) with the mitochondrial aminotransferase which absorbs at 497 mµ. The affinity of the pyridoxal enzyme for this analogue (K = 0.2 mm) is comparable to that of aspartate and is lower than in the supernatant enzyme (K = 0.4 mm). The pyridoxamine form of each isozyme has a higher affinity for erythro-β-hydroxyaspartate than for either glutamate or aspartate. Radioactive exchange studies indicate that transamination can occur between the substrate amino acid and its analogous keto acid. Dissociation constants obtained by this method agree with those of steady state equilibrium spectrophotometric data. A comparison of the intermediates of the mitochondrial and supernatant glutamate-aspartate transaminase indicates that the mitochondrial isozyme has a high affinity for 4-carbon substrates, whereas the supernatant enzyme has no distinctive preference for either 4- or 5-carbon substrates.

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