EPR spectroscopic evidence for the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydratase by coenzyme analogs.

EPR spectra were measured upon incubation of the complex of diol dehydratase with coenzyme analogs in the presence of 1,2-propanediol, a physiological substrate. When the analog in which the D-ribose moiety of the nucleotide loop was replaced by a trimethylene group was used as coenzyme, essentially the same EPR spectrum as that with adenosylcobalamin was obtained. The higher-field doublet and the lower-field broad signals derived from an organic radical and low-spin Co(II) of cob(II)alamin, respectively, were observed. With the imidazolyl counterpart, base-on cob(II)alamin-like species accumulated, but signals due to an organic radical quickly disappeared. When a coenzyme analog lacking the nucleotide moiety was incubated with apoenzyme in the presence of substrate, the EPR spectrum resembling cob(II)inamide was obtained, but no signals due to an organic radical were observed. From these results, it was concluded that the extinction of organic radical intermediates results in inactivation of the enzyme by these coenzyme analogs. Upon suicide inactivation with a [15N2]imidazolyl analog, the octet signals due to Co(II) showed superhyperfine splitting into doublets, indicating axial coordination of 5,6-dimethylbenzimidazole to the cobalamin bound to diol dehydratase.