Nuclear Magnetic Resonance of Aspartate Transaminase: A 19F AND 1H INVESTIGATION OF THE BINDING OF DICARBOXYLIC ACIDS TO VARIOUS FORMS OF EACH ISOENZYME

Abstract

Abstract The binding of succinate and perfluorosuccinate to the supernatant and mitochondrial isoenzymes of aspartate transaminase has been studied using 1H and 19F nuclear magnetic resonance (NMR) as probes. The binding was measured for the pyridoxal (El) and pyridoxamine (Em) forms of the holoenzyme, the apoenzyme, and the pyridoxal form after reduction of the internal Schiff's base with NaBH4 (reduced holoenzyme). The NMR parameters studied were band broadening or chemical shift changes upon dicarboxylic acid binding to the enzyme. The dissociation constant values of succinate and perfluorosuccinate for the active forms of each isozyme, El and Em, are in the 1 to 4 mm range. Kd values of the inactive forms of the supernatant transaminase for perfluorosuccinate are 18.3 mm, reduced form, and 12.0 mm, apoenzyme form. Anions are competitive with dicarboxylic acid binding and, because of mutual exclusion, do not alter the correlation time, τc of bound succinate. This competition allows for the calculation of the chloride affinity of the pyridoxal, Kd = 9 mm (supernatant), 3 mm (mitochondrial), and pyridoxamine forms, Kd = 5 mm (supernatant), 1.6 mm (mitochondrial), of each isoenzyme. Because amino acids and glutarate also compete with perfluorosuccinate binding in the El, Em, and apoenzyme forms, it is concluded that dicarboxylic acids bind to these forms of the enzyme and that amino acids can form complexes with the pyridoxamine form. The correlation time of bound dicarboxylic acid shows an increase, i.e. more restricted segmental motion of succinate, with decreasing pH and is particularly critical in the pH 5 and pH 8 regions. The fastest correlation time of perfluorosuccinate (greatest segmental motion) corresponds to that bound to the reduced holoenzyme. The above effects agree with the competition-pH dependence between dicarboxylic acids and anions. Since there is evidence of the active center histidyl residue as the anion binding site (Cheng, S., and Martinez-Carrion, M. (1972) J. Biol. Chem. 247, 6597–6602) and of the Schiff's base nitrogen as the low pH ligand of a carboxyl group in glutaric acid (Jenkins, W. T., and D'Ari, L. (1966) J. Biol. Chem. 241, 5667), a proposal for the binding of dicarboxylic acids is made. Dicarboxylic acids inhibit because they form a bridge between the active center histidyl residue and the pyridoxal phosphate internal aldimine with the e-amino group of a lysyl residue. Thus, the binding subsites for a competitive inhibitor such as succinate, although structurally similar to the substrates, differ from the productive mode of binding of the amino or keto acids which form covalent enzyme-substrate complexes.