Biophysical techniques

For some time now, our research has concentrated in the area of the chemistry of vitamin B12 and its derivatives, as well as the chemistry of simpler model cobalt chelates capable of stabilizing carbon-cobalt bonds, the unique feature of the biologically active forms of vitamin B12. While the topic of our research is thus bioinorganic (as well as organometallic) in nature, our point of view is decidedly physical organic, as we are interested in the mechanism by which carbon-cobalt bonds are formed and cleaved, the thermodynamics and kinetics of axial ligand substitution pro­cesses, and the association of B12 derivatives with proteins, as well as the mechanisms by which enzymes "activate" the coenzyme form of vitamin B12 by inducing the cleavage of its carbon-cobalt bond. 

One current thrust involves our observations of facile axial ligand diastereomerism on alkylation of cobinamides (B12 derivatives lacking the axial nucleotide) with a variety of alkyl halides. We are studying mechanisms of isomerization and the thermodynamics and kinetics of these reac­tions to try to address the questions of kinetic vs equilibrium control of products. 

We are also studying thermally induced carbon-cobalt cleavage reactions in organocobalt derivatives of vitamin B12. We are trying to understand steric and inductive ef­fects of axial ligands on these reactions as well as the presumably steric basis for the lability of secondary and bulky primary organocobalt derivatives. These experiments are de­signed to provide chemical background for eventual understanding of the enzymatic "activation" of the coenzyme form of vitamin B12. 

Recently, we have developed a force field for the cobalt corrinoids and have used molecular mechanics cal­culations to address important questions of conformation and conformational equilibria in biochemically active derivatives of vitamin B12. Currently, these methods are being advanced by the application of NMR distance restraints to molecular dynamics calculations which will permit the discovery of solution conformations and the three dimensional structures of B12 derivatives which stubbornly refuse to crystallize and cannot be studied by X-ray crystallography. 

We are also currently studying the coenzyme B12-dependent enzyme ribonucleotide reductase (RTPR) from Lactobacillus leichmannii . Recently, stopped-flow kinetic measurements of the "activation" of coenzyme B12 have allowed determination of the activation parameters for the enzyme-induced cleavage of the carbon-cobalt bond. Comparison to the activation parpmeters for the non-enzymatic thermal cleavage of this bond shows that the enzyme lowers the enthalpy of activation by some 15 kcal mol-1 in order to catalyze the reaction. Studies of the NMR properties of 13C and 15N labeled coenzyme B12 with this, and other B12-dependent enzymes are also in progress, along with studies of the structure and enzymology of structural analogs of coenzyme B12, some of which are partially active coenzymes and some of which are inhibitors. 

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