title: Solution structure, enzymatic, and non-enzymatic reactivity of 3-isoadenosylcobalamin, a structural isomer of coenzyme B12 with surprising coenzymic activity.
abstract: The coenzymic activity of eight analogs of coenzyme B(12) (5'-deoxyadenosyl-cobalamin, AdoCbl) with structural alterations in the Ado ligand has been investigated with the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. Six of the analogs were partially active coenzymes, and one, 3-iso-5'-deoxyadenosylcobalamin (3-IsoAdoCbl) was nearly as active as AdoCbl itself. NMR-restrained molecular modeling of 3-IsoAdoCbl revealed a highly conformationally mobile structure which required a four state model to be consistent with the NMR data. Thus, two conformations, one with the IsoAdo ligand over the eastern quadrant of the corrin, and one with the IsoAdo ligand over the northern quadrant, each undergo a facile syn/anti conformational equilibrium in the IsoAdo ligand. Spectrophotometric measurement of the kinetics of RTPR-induced cleavage of the carbon-cobalt bond of 3-IsoAdoCbl showed that it binds to the enzyme with the same affinity as AdoCbl, but its homolysis is only 20% as rapid. Investigation of the non-enzymatic thermolysis of 3-IsoAdoCbl showed that like AdoCbl, 3-IsoAdoCbl decomposes by competing homolytic and heterolytic pathways. A complete temperature-dependent kinetic and product analysis, followed by correction for the base-off species permitted deconvolution of the specific rate constant for both pathways. Eyring plots for the homolysis and heterolysis rate constant cross at 93 degrees C, so that homolysis is the predominant pathway at high temperature, but heterolysis is the predominant pathway at low temperature. At 37 degrees C, the homolysis of 3-IsoAdoCbl is 5.5-fold faster than that of AdoCbl, and the enzyme catalyzes carbon-cobalt bond homolysis in 3-IsoAdoCbl by a factor of 5.9 x 10(7), only 3.9% of the catalytic efficiency with AdoCbl itself. It seems likely that the conformational flexibility of 3-IsoAdoCbl allows it to adopt a coformation in which the hydrogen bonding patterns of the adenine moiety are similar to those of AdoCbl itself, and that this is responsible for the high enzymatic activity of this analog.
authors.affiliation: Department of Chemistry and Biochemistry, Clippinger Laboratories, Ohio University, Athens, OH 45701, USA. brownk3@ohiou.edu
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Brown
authors.forename: Xiang
authors.initials: X
authors.lastname: Zou
authors.forename: Guodong
authors.initials: G
authors.lastname: Chen
authors.forename: Zuping
authors.initials: Z
authors.lastname: Xia
authors.forename: Helder M
authors.initials: HM
authors.lastname: Marques
mesh_terms: D002244:Carbon / Q000737:chemistry; D003035:Cobalt / Q000737:chemistry; D003038:Cobamides / Q000737:chemistry* / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D011408:Propanediol Dehydratase / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012996:Solutions
chemical_list: C490227:3-isoadenosylcobalamin; D003038:Cobamides; D012996:Solutions; D003035:Cobalt; D002244:Carbon; D011408:Propanediol Dehydratase; C000913:cobamamide