Affinity labeling of alanine aminotransferase by 3-chloro-L-alanine.

The pyridoxal form of alanine aminotransferase from pig heart catalyzes the a,/? elimination reaction with 3chloro-L-alanine as the substrate to form equimolar amounts of pyruvate, ammonia, and chloride. The maximum rate of the a,/3 elimination reaction was 2.5 pmol/min/mg at pH 7.0 (25”(Z), approximately 0.5% that of the transamination reaction between L-alanine and 2-oxoglutarate. Time-dependent decrease in the rate of a,/? elimination was found to result from an irreversible inactivation of the enzyme. Michaelis constants for 3chloro-L-alanine were identical in both the a$ elimination and inactivation reactions, indicating that the inactivation occurs via a key intermediate in the a$ elimination reaction. Furthermore, the whole carbon moiety of 3-chloro-L-[U-‘4C]alanine was found to be covalently incorporated into the enzyme protein in a stoichiometric fashion. These observations clearly indicate that chloroalanine acts as an affinity label for alanine aminotransferase as was the case with aspartate aminotransferases (Morino, Y., Osman, A. M., and Okamoto, M. (1974) J. Biol. Chem. 249,6684-6692). The 427 nm band of the native enzyme disappeared instantaneously upon the addition of chloroalanine to form a spectral species absorbing at 325 nm. The pseudo-first order rate constant for this fast process was 53 s-l, which was much greater than that (-5 s-l) for the overall a$ elimination reaction. This fast spectral change was followed by the slow appearance of a new absorption band at 435 nm, which was accompanied by a negative circular dichroic band at 415 nm. The rate of formation of this spectral species (0.36 min-’ at 25°C) coincided exactly with the rate of inactivation of the enzyme. When extrapolated to the infinite concentration of chloroalanine, the rates of inactivation as well as a$ elimination were almost pH-independent. In contrast, the Michaelis constant for chloroalanine varied markedly with the change of pH in both of the a$ elimination and inactivation reactions. The analysis of this pH-dependent variation of Michaelis constants revealed the presence of two ionizable groups on the enzyme with pK, 6.3 and 7.9. Based on these kinetic and spectral data, a possible mechanism of the reaction of alanine aminotransferase with 3-chloro-L-alanine was discussed.