Transient phases of the reaction catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been investigated by stopped-flow visible spectrophotometry and deuterium kinetic isotope effects. The cleavage of adenosylcobalamin (coenzyme B12) to form cob(II)alamin (B12(r)) with ethanolamine as the substrate occurred within the dead time of the instrument whenever coenzyme B12 was preincubated with enzyme prior to mixing with substrate. The rate was, however, slowed sufficiently to be measured with perdeutero ethanolamine as the substrate. Optical spectra indicate that, during the steady states of the reactions with ethanolamine and with S-2-aminopropanol as substrates, approximately 90% of the active sites contain B12(r). Reformation of the carbon-cobalt bond of the cofactor occurs following depletion of substrate in the reaction mixtures, and the rate constant for this process reflects k(cat) of the respective substrates. This late phase of the reaction also exhibits 2H isotope effects similar to those measured for the overall reaction with 2H-labeled substrates. With unlabeled substrates, the rate of cofactor reassembly is independent of the number of substrate molecules turned over in the steady-state phase. However, with 2H-labeled substrates, kinetic isotope effects appear in the reassembly phase, and these isotope effects are maximal after only ~2 equiv of substrate/active site are processed. With 5'-deuterated coenzyme B12 and deuterated substrate, the isotope effect on reassembly is independent of the number of substrate molecules that are turned over. These results indicate that the pool of exchangeable hydrogens in the enzyme-cofactor complex is two a finding consistent with the hydrogens in the C5' methylene of coenzyme B12.
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