Important unanswered questions concerning B12-dependent enzymic processes are: What are the conformational changes which promote the rate of homolytic cleavage of the Co-C bond of coenzyme B12 by a factor of 10-10 and which favor homolytic over heterolytic cleavage in B12 holoenzymes? Why is the Co- N(DMBz) bond so long and what is the role of DMBz in these processes? If the contribution of the enzyme to rearrangement processes (e.g. methylmalonyl-CoA to succinylCoA) is restricted to insuring the existence of long-lived substrate radicals, how do the enzymes prevent radical combination with B12? These and other 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 tightly bound in a protected enzyme pocket inaccessible to probe reagents. Only four alkylcobaltcorrin X-ray structures including coenzyme B12 and methyl B12 have been reported. The molecules are complex and unique and spectroscopic methods are limited by this complexity and by a lack of background data. Therefore, the conformations of well studied coenzyme analogues are unknown. One consequence of this limited information is that there are few clear relationships between structure and spectra. Also, there remains an inadequate background with which to assess the steric factors leading to Co-C bond cleavage. To continue to address these questions, we propose a three-pronged approach. Detailed studies of (i) alkylcobaltcorrins, (ii) synthetic organocobalt species, and (iii) ribonucleotide reductase (RR) are contemplated. The range of conformations possible for alkylcobalamins will be defined via the synthesis of modified or sterically strained alkylcobaltcorrins (cobalamins, cobinamides and other side chain- modified derivatives) and additional organocobalt compounds with sterically or electronically modified equatorial ligands based on the Cosaloph and """"""""Costa"""""""" systems. The effect of structural changes induced in these systems will be assessed by X-ray crystallography, modern spectroscopic methods (CD, 2D and HMQC NMR, Raman) and by reaction rates and mechanistic studies (DMBz dissociation, ligand exchange). Application of some of these methods has produced a wealth of information and provided new insight during this grant period. These methods will be extended to the study of RR. RR is relatively small, with a loosely bound accessible, B12 site and a mechanism of action related to the Fe enzymes. Thus, RR has special advantages and significance. Steric strain in the alkyl linkage between Co and adenine in coenzyme analogues will also be varied and the interaction of the analogues and coenzyme B12 with RR will be investigated. For NMR studies, we will employ cobalamins containing isotopically enriched sites. The spectroscopic (CD, vis, NMR (2D, HMQC)), background to be developed will be generally useful in studies on other B12 holoenzymes and on B12 transport systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029225-08
Application #
3276774
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1980-09-01
Project End
1992-02-29
Budget Start
1989-09-01
Budget End
1992-02-29
Support Year
8
Fiscal Year
1989
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