The coenzyme b12 analog 5'-deoxyadenosylcobinamide-gdp supports catalysis by methylmalonyl-coa mutase in the absence of trans-ligand coordination

J Biol Chem. 2001 Jan 12;276(2):1015-9. doi: 10.1074/jbc.M006842200.

Abstract

Methylmalonyl-CoA mutase is an 5'-adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to cobalt in solution, is replaced by His(610) donated by the active site. The role of the lower axial ligand in the trillion-fold labilization of the upper axial cobalt-carbon bond has been the subject of enduring debate in the model inorganic literature. In this study, we have used a cofactor analog, 5'deoxyadenosylcobinamide GDP (AdoCbi-GDP), which reconstitutes the enzyme in a "histidine-off" form and which allows us to evaluate the contribution of the lower axial ligand to catalysis. The k(cat) for the enzyme in the presence of AdoCbi-GDP is reduced by a factor of 4 compared with the native cofactor AdoCbl. The overall deuterium isotope effect in the presence of AdoCbi-GDP ((D)V = 7.2 +/- 0.8) is comparable with that observed in the presence of AdoCbl (5.0 +/- 0.6) and indicates that the hydrogen transfer steps in this reaction are not significantly affected by the change in coordination state of the bound cofactor. These surprising results are in marked contrast to the effects ascribed to the corresponding lower axial histidine ligands in the cobalamin-dependent enzymes glutamate mutase and methionine synthase.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • Catalysis
  • Cobamides / chemistry*
  • Cobamides / pharmacology*
  • Deuterium
  • Electron Spin Resonance Spectroscopy
  • Guanosine Diphosphate / analogs & derivatives*
  • Guanosine Diphosphate / chemistry*
  • Guanosine Diphosphate / pharmacology*
  • Histidine
  • Kinetics
  • Ligands
  • Methylmalonyl-CoA Mutase / chemistry*
  • Methylmalonyl-CoA Mutase / metabolism*
  • Models, Molecular
  • Molecular Conformation
  • Propionibacterium / enzymology
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Spectrophotometry

Substances

  • Cobamides
  • Ligands
  • Recombinant Proteins
  • guanosine 5'-(5'-deoxyadenosylcobinamide pyrophosphate)
  • Guanosine Diphosphate
  • Histidine
  • Deuterium
  • Methylmalonyl-CoA Mutase
  • cobamamide