Two important unanswered questions concerning B12-dependent enzymic processes are: How does the enzyme promote the homolytic cleavage of the Co-C bond of coenzyme B12? Does the Co play a role in the poorly understood rearrangement reactions such as the conversion of methylmalonyl-CoA to succinyl-CoA by methylmalonyl mutase? These questions have not been fully answered for several reasons. The B12-dependent enzymes are large, making X-ray and some spectroscopic studies difficult. The cofactor generally lies in a protected enzyme pocket inaccessible to probe reagents. Spectroscopic techniques such as CD, vis-UV and EPR which have usually been employed are either limited to paramagnetic species or do not readily yield structural information for such complex molecules. The only reported alkylcobaltcorrin X-ray structure is that of coenzyme B12. One consequence of this limited information is that there is not clear relationship between structure and spectra. Also, there is an inadequate background with which to evaluate the structural feasibility of putative intermediates or to assess the steric factors leading to Co-C bond cleavage. To address these questions, detailed studies of alkylcobaltcorrins and related models are proposed. The range of conformations possible for alkylcobalamins will be defined via the synthesis of sterically strained alkylcobaltcorrins (cobalamins, cobinamides and the heptamethyl ester of cohyrinic acid) and models compounds (such as Cosaloph and """"""""Costa"""""""" model compounds). The effect of structural changes induced in these systems will be assessed by X-ray crystallography, spectroscopy (CD, NMR, Raman) and by reaction rates and mechanistic studies (Co-C bond cleavage, ligand exchange, model rearrangement reactions). Changes are anticipated in the Co-to-axial ligating atom bond lengths, the Co position relative to corrin, corrin side chains, etc. The structure of the prototype alkylcobalamin, methylB12, is nearing completion. Steric strain in the alkyl linkage between Co and adenine in cobalamin analogues will also be varied and the interaction of the analogues with B12-dependent enzymes such as ethanolamine ammonia lyase will be investigated. With the spectroscopic background developed, CD, vis, NMR, and Raman studies will be initiated on B12 holoenzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029225-04
Application #
3276771
Study Section
(SSS)
Project Start
1980-09-01
Project End
1987-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
4
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Emory University
Department
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Shiang, Joseph J; Cole, Allwyn G; Sension, Roseanne J et al. (2006) Ultrafast excited-state dynamics in vitamin B12 and related cob(III)alamins. J Am Chem Soc 128:801-8
Calafat, A M; Taoka, S; Puckett Jr, J M et al. (1995) Structural and electronic similarity but functional difference in methylmalonyl-CoA mutase between coenzyme B12 and the analog 2',5'-dideoxyadenosylcobalamin. Biochemistry 34:14125-30
Pagano, T G; Marzilli, L G (1989) Vitamin B12 monocarboxylic acids: unambiguous isomer assignments by modern two-dimensional NMR spectroscopy. Biochemistry 28:7213-23