Mechanism-based inactivation of pig heart L-alanine transaminase by L-propargylglycine. Half-site reactivity.

L-Alanine transaminase (EC 2.6.1.2) from pig heart was found to be a dimer, with a subunit molecular weight of 55,000 and one pyridoxal phosphate bound/subunit. Seven free sulfhydryl groups/subunit were detected. Isoelectric focusing revealed three species (native pI values, 5.3 to 5.5). L-Propargylglycine was found to inactivate the enzyme at 37 degrees C with a KI = 3.9 mM and an observed maximal first order rate constant, kinact = 0.26 min-1. Incorporation of 1 [14C]propargylglycine molecule/dimer leads to greater than 97% inactivation, suggesting half-site reactivity, while the unalkylated subunit is still apparently capable of processing L-alanine, L-propargylglycine, and beta-chloro-L-alanine. The minimal stoichiometric ratio necessary for inactivation was determined to be 2.7 L-propargylglycine molecules/enzyme subunit, 2.2 molecules/subunit undergoing transamination before inactivation ensues. A deuterium kinetic isotope effect of 3.5 was observed for inactivation with DL-[2-2H]propargylglycine.