In the presence of the aldimine form of aspartate aminotransferase, L-serine O-sulfate undergoes a β-elimination reaction leading to the formation of equimolar amounts of pyruvate, protons, ammonium, and sulfate ions. The reaction was followed by measuring either proton release in a pH-Stat or pyruvate production by a coupled reduced nicotinamide- adenine dinucleotide dependent reducing system. While the initial rates determined by the two methods are identical, the progress curves, though both biphasic, are quite different. This difference is explained by a slow transamination between L-serine O-sulfate and the aldimine enzyme to give the aminic enzyme, which is unable to catalyze the β-elimination reaction. Pyruvate and other keto acids, by transaminating with the aminic enzyme, regenerate the aldimine enzyme and thus maintain the rate of β elimination at a higher level. Spectrofluorimetric evidence indicates that transamination does occur between L-serine O-sulfate and pyridoxal phosphate in the presence of aspartate aminotransferase. In addition, an irreversible reaction takes place between this substrate and aspartate aminotransferase resulting in complete inactivation of the enzyme. Understanding of the various events occurring in the system has allowed the determination of the steady-state kinetic parameters for the β-elimination reaction. At pH 6.5 and 25°, Km was 7 × 10-2 m and kcat = 12 sec-1. Transamination proceeds much more slowly. These findings show that aspartate aminotransferase is capable of catalyzing, on the same substrate, two simultaneous reactions; the progress of one of these reactions prevents the other from occurring. The irreversible inactivation of the enzyme by a substrate analog provides a way of specifically labeling the active site. © 1969, American Chemical Society. All rights reserved.
