Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B₁₂‐dependent diol dehydratase and ethanolamine ammonia‐lyase

Abstract

AdoCbl participates as coenzyme for the enzymes that catalyze carbon skeleton rearrangements, heteroatom eliminations, and intramolecular amino group migrations [1][2][3]. For example, diol dehydratase (EC 4.2.1.28) and ethanolamine ammonia-lyase (EC 4.3.1.7) catalyze the dehydration of 1,2-diols and the deamination of ethanolamine to the corresponding aldehydes, respectively [4][5][6]. These reactions proceed by a radical mechanism, and an essential early event in all the AdoCbl-dependent rearrangements is the generation of a catalytic radical (adenosyl radical) by homolytic cleavage of the coenzyme's Co-C bond [1,7].Recently, the X-ray structures of several AdoCbldependent enzymes have been solved in complexes with cobalamins [8][9][10][11][12][13]. Spatial isolation of the radical intermediates in the active site cavity seems to be the common strategy for the so-called 'negative catalysis' of the Re´tey's concept [14]. The interactions of the coenzyme's adenine moiety with enzymes were revealed with methylmalonyl-CoA mutase [15,16] [x-(Adenosyl)alkyl]cobalamins (homoadenosylcobalamins) are useful analogues of adenosylcobalamin to get information about the distance between Co and C5¢, which is critical for Co-C bond activation. In order to use them as probes for exploring the active sites of enzymes, the coenzymic properties of homoadenosylcobalamins for diol dehydratase and ethanolamine ammonia-lyase were investigated. The k cat and k cat ⁄ K m values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regular coenzyme with diol dehydratase, respectively. The k cat ⁄ k inact value showed that the holoenzyme with this analogue becomes inactivated on average after about 3000 catalytic turnovers, indicating that the probability of inactivation during catalysis is almost 500 times higher than that for the regular holoenzyme. The k cat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonialyase, as judged from the initial velocity, but the holoenzyme with this analogue underwent inactivation after on average about 50 catalytic turnovers. This probability of inactivation is 3800 times higher than that for the regular holoenzyme. When estimated from the spectra of reacting holoenzymes, the steady state concentration of cob(II)alamin intermediate from adenosylmethylcobalamin was very low with either diol dehydratase or ethanolamine ammonia-lyase, which is consistent with its extremely low coenzymic activity. In contrast, neither adenosylethylcobalamin nor adeninylpentylcobalamin served as active coenzyme for either enzyme and did not undergo Co-C bond cleavage upon binding to apoenzymes. AbbreviationsAdoCbl, adenosylcobalamin or coenzyme B 12 ; AdoEtCbl, adenosylethylcobalamin; AdoMeCbl, adenosylmethylcobalamin or homocoenzyme B 12 ; AdePeCbl, adeninylpentylcobalamin.

Cofactor Selectivity in Methylmalonyl Coenzyme A Mutase, a Model Cobamide-Dependent Enzyme

“…Among the cobamides we tested, the maximum differences in Sm MCM turnover were 3-fold. We did not observe inhibition of MCM activity with any cobamides, in contrast to the strong inhibition that has been observed with analogs containing variations in the upper ligand or central metal, known as antivitamins (111–113).…”

Cofactor selectivity in methylmalonyl-CoA mutase, a model cobamide-dependent enzyme

“…Among the cobamides we tested, the maximum differences in SmMCM turnover were 3-fold. We did not observe inhibition of MCM activity with any cobamides, in contrast to the strong inhibition that has been observed with analogs containing variations in the upper ligand or central metal, known as antivitamins (Calafat et al, 1995;Fukuoka et al, 2005;Widner et al, 2016).…”

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12‐Dependent Diol Dehydratase and Ethanolamine Ammonia‐Lyase

“…The observed small activity of AdoMeCbl as cofactor of DD and EAL, that is, 0.27 % and 0.13 % that of AdoCbl for DD and EAL, respectively, [15] has been a puzzle that induced us to undertake the present crystallographic study. It is highly improbable that these residual activities are caused by AdoCbl as an impurity.…”

“…Thus, these homologues of coenzyme B 12 also seem to be useful probes for exploring the active sites of AdoCbl‐dependent enzymes. Spectroscopic studies indicated that these analogues do not undergo significant Co−C bond cleavage upon binding to apoenzymes within at least 10 min [6a,15] …”