Project Details
Description
DESCRIPTION (Adapted from abstract): Diseases in humans that are caused by
deficiencies or malabsorption of vitamin B12 include hyperhomocysteinemia,
megaloblastic anemia, pernicious anemia and methylmalonic acidemia.
Adenosylcobalamin (Coenzyme B12)-dependent enzymes will be studied using
multifrequency EPR, ESEEM (electron spin echo envelope modulation), and ENDOR
(electron nuclear double resonance) in the low frequency (4-18 GHz) and high
frequency (140 GHz) regimes with the ultimate goal of catalytic mechanism
determination. Of particular interest will be the exchange-coupled
cob(ll)alamin-organic radical pairs generated as intermediates in the catalytic
cycles of glutamate mutase from Clostridium cochlearium and ribonucleoside
triphosphate reductase (RTPR) from Lactobacillus leichmannIi. Model cobalt(ll)
compounds, including cob(ll)alamin and Co(ll)-bis(dimethylglyoximes) will also
be studied to provide insight into the spectroscopic characteristics and
structure of the cobalamin which is involved in exchange-coupled pairs in the
enzymes. For the case of glutamate mutase, selectively isotopically labeled
substrates (glutamates) are available (2H, 13C and 15N) to aid in the
structural identification. For RTPR, incubation with mechanistic inhibitors
produces a rich variety of EPR spectra. Identification of these radical species
will provide insight into the mechanism of inhibition and perhaps of catalysis.
Overall goals of this project include: 1) determination of structural details
of the enzyme-bound cob(ll)alamin species in the exchange-coupled pair, through
equatorial and axial nitrogen hyperfine and quadrupole parameters; 2)
investigation of the remote nitrogen of the axially coordinated base I (either
histidine or dimethylbenzimidazole) in both the enzyme-bound cob(ll)alamin and
exchange-coupled pair; 1 3) identification and structural characterization of
the radical species coupled to the enzyme-bound cob(ll)alamin; 4) precise
determination of the electron-electron exchange and zero field splitting
(dipolar) interaction to establish the distance between and relative
orientation of the two paramagnetic species. Achievement of these aims is
necessary to give a complete picture of the catalytic mechanisms of these
enzymes, and may give insight into the role of the enzyme in selectively
increasing homolytic reactivity of the adenosylcobalamin carbon-cobalt bond by
a factor of about 1 x 10(12) relative to free adenosylcobalamin. This "enzyme
activation" of the cobalt-carbon bond j is a requirement for
adenosylcobalamin-dependent catalysis.
Status | Finished |
---|---|
Effective start/end date | 2/1/00 → 1/31/06 |
ASJC
- Catalysis
- Medicine(all)
- Biochemistry, Genetics and Molecular Biology(all)
Fingerprint
Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.