Efects of Oxaloacetate and l‐Glutamate on Glyceraldehyde 3‐Phosphate: Inhibition of Aspartate Aminotransferase Isozymes as Measured by a 2‐Oxoglutarate Dehydrogenase Coupled Assay

A new coupled reaction for the measurement of aspartate aminotransferase activity with oxaloacetate and l‐glutamate as substrates is described. This reaction measures the appearance of the product, 2‐oxoglutarate, with 2‐oxoglutarate dehydrogenase. It avoids the high concentrations of phosphate or arsenate which have been used to catalyze tautomerization in those assays which utilize the absorbance of the enol form of oxaloacetate to measure its concentration. These salts inhibit the cationic isozyme of aspartate aminotransferase. Values for K_m(oxaloacetate) and K_m(l‐glutamate) were 0.037 mM and 7.8 mM, respectively, for rat liver anionic isozyme and 0.006 mM and 8.6 mM, respectively, for the cationic isozyme. Glyceraldehyde 3‐phosphate inhibition of the anionic isozyme was partially competitivepartially noncompetitive with respect to oxaloacetate and partialy noncompetitive with respect to l‐glutamate, with β= 0.31. Inhibition by glyceraldehyde‐3‐P of the cationic isozyme was non‐competitive with respect to l‐glutamate. K_i values for glyceraldehyde‐3‐P for the anionic isozyme were 0.57 mM in the presence of l‐glutamate and 1.5 mM in the presence of oxaloacetate. The K_i value for the cationic isozyme in the presence of l‐glutamate was 0.11 mM. These values are in agreement with those that were reported previously for inhibition in the presence of l‐aspartate and 2‐oxoglutarate. The similarity between the types of inhibition in both directions of transamination indicates that both keto acid substrates bind to the isozymes at a single site and compete with glyceraldehyde‐3‐P for that site. The noncompetitive nature of inhibition in the presence of amino acid substrates suggests that at least one point of their attachment is outside of the site to which the keto acids and glyceraldehyde‐3‐P bind.