title: Isotope effects for deuterium transfer between substrate and coenzyme in adenosylcobalamin-dependent glutamate mutase.
abstract: A key step in the mechanism of all adenosylcobalamin-dependent enzymes is the abstraction of a hydrogen atom from the substrate by a 5'-deoxyadenosyl radical generated by homolytic fission of the coenzyme cobalt-carbon bond. We have investigated the isotope effects associated with this process for glutamate mutase reacting with deuterated glutamate. The kinetics of deuterium incorporation into 5'-deoxyadenosine (5'-dA) during the reaction were followed by rapid chemical quench, using HPLC and electrospray mass spectrometry to analyze the 5'-dA formed. The kinetics of 5'-dA formation are biphasic, comprising a rapid phase k(app) = 37 +/- 3 s(-)(1) and a slower phase k(app) = 0.9 +/- 0.4 s(-)(1). The mass spectral data clearly show that the faster phase is associated with the formation of monodeuterated 5'-dA whereas the slower phase is associated with the incorporation of a second and then a third deuterium into 5'-dA. This observation implies that a large inverse equilibrium secondary isotope effect is associated with the formation of 5'-dA from adenosylcobalamin. The primary deuterium kinetic isotope effects on V and V/K for the formation of 5'-dA were determined from time-based and competition experiments. (D)V = 2.4 +/-0.4 whereas (D)(V/K) = 10 +/- 0.4, implying that an isotopically insensitive step is partially rate-determining. The additional data provided by these experiments cause us to revise our interpretation of earlier UV-visible stopped-flow kinetic measurements of AdoCbl homolysis obtained with deuterated substrates.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
authors.forename: Mou-Chi
authors.initials: MC
authors.lastname: Cheng
authors.forename: E Neil G
authors.initials: EN
authors.lastname: Marsh
mesh_terms: D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003038:Cobamides / Q000737:chemistry* / Q000378:metabolism*; D003839:Deoxyadenosines / Q000378:metabolism; D003903:Deuterium / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods; D004735:Energy Transfer; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D003038:Cobamides; D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D005609:Free Radicals; D020035:Holoenzymes; D018698:Glutamic Acid; C033714:5'-deoxyadenosine; D006859:Hydrogen; D003903:Deuterium; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000913:cobamamide