The broad and long-term objectives of this research are to provide a molecular level understanding of the family of adenosylcobalamin (Coenzyme B 12)-dependent and related enzymes that catalyze C-H bond activations, followed by rearrangement or reduction reactions, in nature. This family of enzymes includes those with B 12, Fe2, Mn2 (and probably other, yet to be discovered) active sites. Recent evidence suggests that these enzymes use an unusual, and still poorly understood, radical chain mechanism of action; the wide distribution of these enzymes in bacteria, plants, and mammals including humans attests to the importance of this radical chain mechanism. The Coenzyme B12 cofactor is essential for the normal maturation of erythrocytes; in man, insufficient B12 leads to pernicious anemia and clinical features that include megaloblastic anemia, malignant neoplasms, and neurological disorders. More specifically, adenosylcobalamin is an essential cofactor for methylmalonyl-CoA mutase and methylcobalamin is an essential cofactor for homocysteine methyltransferase.
The specific aims outlined are designed to provide a chemical paradigm for the individual, elementary mechanistic steps thought to operate in the Coenzyme B12-initiated radical chain reaction. Furthermore, one of the steps, the initial homolysis of Coenzyme B12's Co-C bond, is subject to an unusually large, 1012, enzymic rate enhancement. Understanding this enormous effect, in this perhaps prototype system, is another focus of these studies. Lastly, studies are outlined that involve a central Coenzyme B12 With one or two covalently linked polypeptide a-helices. This work, which takes advantage of the nearly ideal features of the B12 cofactor, begins to probe the very important and general but ill-understood question of how polypeptide and protein low frequency motions couple to an enzyme catalysis reaction coordinate, such as the simple (and thus nearly ideal) Co---C cleavage reaction coordinate. Such protein-assisted dynamics are essential components of the enzymic transformations that sustain living organisms, yet remain poorly understood.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK026214-12
Application #
3227781
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1979-07-01
Project End
1994-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
12
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Schools of Arts and Sciences
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
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Doll, Kenneth M; Fleming, Paul E; Finke, Richard G (2002) The synthesis and characterization of 8-methoxy-5'-deoxyadenosylcobalamin: a coenzyme B(12) analog which, following Co-C bond homolysis, avoids cyclization of the 8-methoxy-5'-deoxyadenosyl radical. J Inorg Biochem 91:388-97
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Brasch, N E; Finke, R G (1999) A simple, convenient and direct method for assessing the purity of cobalamins. J Inorg Biochem 73:215-9
Suto, R K; Whalen, M A; Bender, B R et al. (1998) Synthesis of gamma-phosphate-linked nucleoside affinity chromatography resins for protein purification, including ribonucleoside triphosphate reductase. Nucleosides Nucleotides 17:1453-71
Finke, R G; Martin, B D (1990) Coenzyme AdoB12 vs AdoB12.-homolytic Co-C cleavage following electron transfer: a rate enhancement greater than or equal to 10(12). J Inorg Biochem 40:19-22