Isotope effects in the transient phases of the reaction catalyzed by ethanolamine ammonia-lyase: determination of the number of exchangeable hydrogens in the enzyme-cofactor complex.
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 B(12)) to form cob(II)alamin (B(12r)) with ethanolamine as the substrate occurred within the dead time of the instrument whenever coenzyme B(12) 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 B(12r). 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 (2)H isotope effects similar to those measured for the overall reaction with (2)H-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 (2)H-labeled substrates, kinetic isotope effects appear in the reassembly phase, and these isotope effects are maximal after only approximately 2 equiv of substrate/active site are processed. With 5'-deuterated coenzyme B(12) 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 B(12).
Bandarian
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Department of Biochemistry, University of Wisconsin-Madison, 1710 University Avenue, Madison, Wisconsin 53705, USA.
Reed
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D002384:Catalysis; D003038:Cobamides / Q000737:chemistry / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D004981:Ethanolamine Ammonia-Lyase / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen; D007700:Kinetics; D008956:Models, Chemical; D013053:Spectrophotometry; D013379:Substrate Specificity; D014316:Tritium
D003038:Cobamides; D003067:Coenzymes; D014316:Tritium; D006859:Hydrogen; D003903:Deuterium; D004981:Ethanolamine Ammonia-Lyase; C000913:cobamamide