Synthesis and Catalytic Activity of Spin-Labeled Cobinamide Coenzymes

P. Y. Law, Dennis G. Brown, E. L. Lien, Bernard M. Babior, J. M. Wood

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37 Scopus citations


In order to facilitate the study of the mechanism of action of corrin-dependent enzymes, the preparation of active spin-labeled cobinamides was undertaken. The synthesis of these compounds has been accomplished by the coordination of a piperidine-N-oxyl stable free radical to the cobalt atom of cobinamides through the oxygen atom of the TV-oxyl functional group. By this procedure spin-labeled derivatives of methylcobinamide (MCN) and 5'-deoxyadenosylcobinamide (DACN) are obtained in good yield. The synthesis of DACN was accomplished as a result of our development of a new procedure for the synthesis of 5'-deoxyadensyIcobinamide (DAC) in good yields. The structure of these derivatives has been rigorously defined by 220-MHz proton nuclear magnetic resonance, by electron spin resonance, by circular dichroism, and by the determination of pKa values for displacement of the nitroxide ligand. Dissociation constants for these coordination complexes have been determined for the pH at which enzyme assays were performed (i.e., 7.5). In the enzymatic experiments 4-hydroxy-2,2,6,6-tetramethylpiperidine-Ar-oxyl 5'-deoxyadensyIcobinamide (DACN) was found to replace 5'-deoxyadenosylcobalamin (DMBC) in ethanolamine am-monia-lyase. Electron spin resonance has been used to demonstrate that homolytic cleavage of the Co-C σ bond occuss when ethanolamine is added to the spin-labeled enzyme-DACN complex. Control experiments involving photolysis of the spin-labeled corrinoid have been performed to help confirm this mechanism. Homolysis of the Co-C σ bond also occuss when acetaldehyde and ammonium ions are added to the enzyme-DACN complex. This experiment suggests that partial reversibility of this deamination reaction can take place.

Original languageEnglish (US)
Pages (from-to)3428-3435
Number of pages8
Issue number18
StatePublished - Aug 1 1971


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