Chloride Affinity Research Articles

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

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.

Catalysis by metal cations. Part II. A further study of the elimination of chloride ion from chloro(ethylenediaminetriacetatoacetate)cobaltate(III) catalysed by metal cations

An earlier study of the kinetics of the elimination of chloride ion from chloro(ethylenediaminetriacetatoacetate)cobaltate(III) catalysed by bivalent cations has been extended to include tervalent cations. The equilibrium constants for the association between the substrate and several of the tervalent catalysts can be obtained from the kinetic measurements. The reactivity of a catalyst is strongly dependent upon its affinity for chloride and for the carboxylate group, but the retention or otherwise of the primary co-ordination sphere of the catalyst in its interaction with the substrate is also important.

Effects of halides on binding of trace concentrations of mercury to human erythrocytes.

Summary. Trace concentrations of mercury (in the range of 10‐‐6 M) in the presence of 0.144 M‐NaCl or NaBr bind to erythrocytes to a limited degree, whereas mercury in the presence of 0.144 M‐NaI under similar conditions is completely bound by erythrocytes. The limited binding of mercury to erythrocytes in the presence of bromide and chloride is due to the presence of substances in the incubation fluid that bind mercury. Little of the mercury in iodide solution is bound by these extracellular substances, whereas most of the mercury in bromide and chloride solution is bound to these substances. The erythrocyte membrane appears to function like a dialysis membrane by permitting the unbound fraction (small mercury‐halide complexes) to enter erythrocytes for intracellular binding while excluding the bound fraction of mercury (large molecules). The high concentration of binding substances in the erythrocytes results in binding of mercury that would not bind to dilute extracellular substances. Cellulose dialysis tubing mimics many of the properties of the erythrocyte system because of the presence of binding sites for mercury on the tubing. The data are best explained by the increasing affinity of chloride, bromide and iodide ions for mercury and the reciprocally related ease with which halides can be displaced from mercury‐halide complexes to form ‘active’ positively charged mercury ions for binding to receptor sites. The factors demonstrated in this system may be relevant to the accumulation of other metals within cells.