My long-term goals are to develop insightful and informative spectroscopic methods for assessing the dynamic processes involving cobamide cofactors (Cbas) bound to enzymes or transport proteins and to elucidate the fundamental chemistry relevant to function. Conformational changes in Cbas induced through cofactor-protein contacts and protein conformational changes are important in enzyme function. To achieve these goals, studies are proposed with enzymes, with derivatives of natural B12 compounds, and with models, including a novel pair of lariat-type models recently synthesized in my laboratory. Our focus has been on near-IR FT-Raman and on NMR spectroscopic methods. The progress we have made recently has applications to enzymes dependent on one of the Cbas, 5'- deoxyadenosylcobalamin (AdoCbl) or methylcobalamin (MeCbl). I conclude that during the requested renewal period our expertise in spectroscopy and model chemistry would be best applied to MeCbl-dependent methionine synthase (MS) and to elucidation of the interaction of thiols/thiolates with both model and B12 species. The MeCbl-binding domain of 133-kDa MS is included in a relatively small (<28 kDa) domain, which was recently crystallized. Such domain-proteins should give high-quality spectra, and it is possible that even smaller domain-proteins will be identified by the alternative methods of PCR-cloning-expression or by proteolytic digestion. Greater knowledge of MeCbl binding is essential in order to eventually understand MS function. Several approaches are proposed to relate the binding in the more readily characterized domain-proteins to that in the MS holoenzyme. These approaches include FT-Raman, CD and 31P and 19F NMR, photolysis of the bound Cbl analogues, spin labels, propionamide-modified analogues, and novel cleaving/probe analogues. Results on both domains and holoenzyme will be compared. Spectroscopic and chemical studies of Cbl- and Cbl-analogue-binding to these domains should allow the development of methods for examining the more complex and larger AdoCbl-dependent enzymes. The involvement of thiols or thiolates in MS function has one clear and another suspected, unknown role. The clear role involves the substrate, homocysteine, which is methylated by MeCbl. A cysteine, (probably C772, the only cysteine in the binding domain), is protected from thiol reagents by Cbl binding. This observation, along with evidence that cysteines are essential in many AdoCbl-dependent enzymes, implicates a cysteine in MS function. Furthermore, by some isolation procedures, sulfitoCbl was isolated from MS. Finally, the form of B12 that is the precursor to coenzyme formation is glutathioneCbl. There is a clear need to understand thiol/thiolate-Cbl interactions. However, our knowledge of thiol/thiolate organocobalt chemistry is rudimentary; the few published studies are contradictory and complex. The high air sensitivity of reduced B12 and model species, combined with the normal difficulties of thiol/thiolate redox transition metal chemistry, has retarded progress in this field. Numerous experiments are proposed with Cbl-derivatives and synthetic models to elucidate this fundamental organocobalt chemistry.
| 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 |
