Glutamic-aspartic Transaminase. 8. Equilibrium Kinetics with Aspartate.

Pig heart “soluble” glutamic-aspartic transaminase (L-aspartate : 2-oxoglutarate aminotransferase, EC 2.6.1.1) will catalyze the exchange of an amino group between glutamate and ketoglutarate without the participation of any other amino acid or keto acid (1). This “exchange transamination” also occurs between aspartate and oxaloacetate. The rates of these reactions are comparable to the physiological reversible transfer from aspartate to ketoglutarate under identical conditions. Because the concentrations of substrates do not change, such “exchange transaminations” provide ideal steady state systems for the spectroscopic analysis of enzyme substrate complexes when the enzyme concentration is greatly increased. Two forms of the enzyme, (El) and (Ez), have been prepared (2-5) which contain pyridoxal phosphate and pyridoxamine phosphate, respectively. Since these two forms of the enzyme are instantaneously, and specifically, interconverted by the addition of amino acid and keto acid substrates, they must be considered to be among the components of the exchange transamination equilibria (Fig. 1). Although the pyridoxal and pyridoxamine forms of the enzyme have been well characterized spectroscopically, rigorous characterization of the other complexes in the equilibria has not yet been accomplished, owing to the fact that a wide variety of such complexes exists and their spectra closely resemble those of the pyridoxal and pyridoxamine forms of the enzyme (1, 2, 6). Until now this has prevented the determination of the relative stabilities of the enzyme substrate complexes and hence the elucidation of the detailed mechanism of the reaction. Although exchange transamination at a constant pH between amino acid (A) and keto acid (0) involves many forms of the enzyme, these are equilibrium mixtures of relatively few possible species as shown in Fig. 1. Three possible types of binary complexes are shown in Fig. 1. The complex (0 .E1) between keto acid (0) and the pyridoxal form of the enzyme (El) together with that (A &) between amino acid (A) and the pyridoxamine form of the enzyme (&) we term “abortive complexes.” This term has been used to describe ternary complexes with lactic dehydrogenase which contain either lactate and reduced diphosphopyridine nucleotide or pyruvate and diphosphopyridine nucleotide. We refer to (EX) as the intermediary binary complex because it appears to be an obligatory intermediate on the