ROLE OF PERIPHERAL SIDE-CHAINS OF VITAMIN-B12 COENZYMES IN THE REACTION CATALYZED BY DIOLDEHYDRASE

Isomers of adenosylcobalamin in which one of the three amide groups of the propionamide side chain of the corrin ring was converted to-COOH, -COOCH3, or-CONHCH3 were tested for coenzyme activity with dioldehydrase. The coenzyme activity of these nine isomers ranged from 86 to 7% that of the normal coenzyme. The rate-limiting step with the monocarboxylic acid analogues was the same as with the normal coenzyme as indicated by a 12-fold deuterium isotope effect on the maximal velocity of the reaction with [1,1-2H]-l,2-propanediol. With the b site modifications of the coenzyme, the maximal velocities decrease in the order -COO ≫ CONHCH3, COOCH3 suggesting inhibition by steric effects, while at the diagonal e site the maximal velocities decrease in the order -CONHCHj, -COO-, -COOCH3 suggesting the importance of H-bond donation from coenzyme to enzyme. The binding of the adenosyl and cyano forms of the analogues to apoenzyme was reversible, although the hydroxo forms bind essentially irreversibly. This is in contrast to the adenosylcobalamin and cyanocobalamin which bind essentially irreversibly. In the presence of propanediol, the e-carboxylic acid, e-methyl ester, and b- and e-methylamide coenzymes brought about irreversible inactivation of the enzyme. Optical and EPR spectroscopy demonstrated that except for the complexes with the inactivating analogues, the holoenzymes of the other analogues generate an organic radical and cob (II) alamin during catalysis, whose steady-state concentrations correlated with their activity. The concentrations of the cob(II)alamin species and the radical species are equal. EPR spectra of the complexes with the four inactivating analogues show the accumulation of a disproportionally higher concentration of cob (II) alamin relative to the organic radical. These findings indicate the quenching of the organic radical intermediates in side reactions presumably due to their improper positioning. All of the four binary complexes of the inactivating analogues with the apoenzyme were relatively stable in the absence of substrate. In contrast, the complexes with the normal coenzyme and with the analogues which do not inactivate are unstable in the absence of substrate, presumably due to reaction of the radical species with 02. This stability of the inactivating complexes suggests insufficient activation of the coenzyme, i. e., lower levels of the dissociated form. Since the modifications of the propionamide side chains used do not affect the intrinsic chemical properties of the coenzyme, all of the observed structural and kinetic effects result from alterations of the coenzyme-protein interaction. The present data thus indicate that the interactions of the coenzyme propionamide side chains with the apoprotein facilitate the homolytic cleavage of the C-Co bond and contribute to the stabilization of the radical intermediates. © 1979, American Chemical Society. All rights reserved.