title: Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase.
abstract: Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and product in the reaction catalyzed by glutamate mutase. The isotope partitions between glutamate and methylaspartate in nearly 1:1 ratio, regardless of the direction in which the overall reaction is proceeding. This is consistent with a free-energy profile in which the interconversion of the intermediate glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product. Initial velocity measurements have been used to measure the tritium isotope effects for the transfer of tritium from adenosylcobalamin to product in each direction. The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate. The large magnitude of these isotope effects makes it likely that the rate-determining step may be altered by the substitution of tritium for hydrogen in the reaction. The results of these experiments are compared with previous isotope effect measurements made on other adenosylcobalamin-dependent enzymes.
authors.affiliation: Department of Chemistry, University of Michigan, 930 N. University, Ann Arbor, Michigan 48109-1055, USA.
authors.forename: H W
authors.initials: HW
authors.lastname: Chih
authors.forename: E N
authors.initials: EN
authors.lastname: Marsh
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003038:Cobamides / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D018698:Glutamic Acid / Q000737:chemistry*; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000378:metabolism; D007554:Isotopes / Q000737:chemistry*; D007700:Kinetics; D016202:N-Methylaspartate / Q000737:chemistry; D011485:Protein Binding; D013816:Thermodynamics; D013997:Time Factors; D014305:Triose-Phosphate Isomerase / Q000737:chemistry; D014316:Tritium / Q000737:chemistry*
chemical_list: D003038:Cobamides; D007554:Isotopes; D014316:Tritium; D018698:Glutamic Acid; D016202:N-Methylaspartate; D014305:Triose-Phosphate Isomerase; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000913:cobamamide