Coenzyme B12 dependent diol dehydratase undergoes mechanism‐based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co–C bond. Bachovchin et al. [Biochemistry16, 1082–1092 (1977)] reported that glycerol bound in the GS conformation, in which the pro‐S‐CH2OH group is oriented to the hydrogen‐abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X‐ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)‐1,2‐propanediol, i.e. in the GS conformation, with its 3‐OH group hydrogen bonded to Serα301, but not to nearby Glnα336. kinact of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild‐type enzyme. kcat/kinact showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild‐type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)‐1,2‐propanediol decreased on these mutations. The substrate activities towards longer chain 1,2‐diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2‐diols. Database Structural data are available in the Protein Data Bank under the accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3AUJ. Structured digital abstract  http://www.uniprot.org/uniprot/Q59472, http://www.uniprot.org/uniprot/Q59471 and http://www.uniprot.org/uniprot/Q59470 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8301985)

Adenosylcobalamin-dependent diol dehydratase of (1). This causes 60°clockwise and 70°counterclockwise rotations of the C(1)-C(2) bond of the (R)-and (S)-isomers, respectively, if viewed from K؉ . A modeling study of 1,1-gem-diol intermediates indicated that new radical center C(2) becomes close to the methyl group of 5-deoxyadenosine. Thus, the hydrogen back-abstraction (recombination) from 5-deoxyadenosine by the product radical is structurally feasible. It was also predictable that the substitution of the migrating hydroxyl group by a hydrogen atom from 5-deoxyadenosine takes place with the inversion of the configuration at C(2) of the substrate. Stereospecific dehydration of the 1,1-gem-diol intermediates can also be rationalized by assuming that Asp-␣335 and Glu-␣170 function as base catalysts in the dehydration of the (R)-and (S)-isomers, respectively. The structure-based mechanism and stereochemical courses of the reaction are proposed.

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.

The roles of the D-ribosyl moiety and the bulky axial ligand of the nucleotide loop of adenosylcobalamin in coenzymic function have been investigated using two series of coenzyme analogs bearing various artificial bases. The 2-methylbenzimidazolyl trimethylene analog that exists exclusively in the base-off form was a totally inactive coenzyme for diol dehydratase and served as a competitive inhibitor. The benzimidazolyl trimethylene analog and the benzimidazolylcobamide coenzyme were highly active for diol dehydratase and ethanolamine ammonia-lyase. The imidazolylcobamide coenzyme was 59 and 9% as active as the normal coenzyme for diol dehydratase and ethanolamine ammonia-lyase, respectively. The latter analog served as an effective suicide coenzyme for both enzymes, although the partition ratio (k(cat)/k(inact)) of 630 for ethanolamine ammonia-lyase is much lower than that for diol dehydratase. Suicide inactivation was accompanied by the accumulation of a cob(II)amide species, indicating irreversible cleavage of the coenzyme Co-C bond during the inactivation. It was thus concluded that the bulkiness of a Co-coordinating base of the nucleotide loop is essential for both the initial activity and continuous catalytic turnovers. Since the k(cat)/k(inact) value for the imidazolylcobamide in diol dehydratase was 27-times higher than that for the imidazolyl trimethylene analog, it is clear that the ribosyl moiety protects the reaction intermediates from suicide inactivation. Stopped-flow measurements indicated that the rate of Co-C bond homolysis is essentially unaffected by the bulkiness of the Co-coordinating base for diol dehydratase. Thus, it seems unlikely that the Co-C bond is labilized through a ground state mechanochemical triggering mechanism in diol dehydratase.

Cytotoxicities of propranolol (PL) and its active metabolite, 4-hydroxypropranolol (4-OH-PL), were examined in a human hepatoma cell line, Hep G2. Hep G2 cells were cultured in the presence of β-naphthoflavone (BNF, 25 or 50 µM), lansoprazole (LPZ, 25 or 50 µM) or 0.5% dimethylsulfoxide (vehicle) for 48 hr. The cells were harvested, and microsomal and cytosolic fractions were prepared by differential centrifugation methods. Various enzyme activities were determined as follows: microsomal 7-ethoxyresorufin (ER) O-deethylation as a CYP1A1 index, microsomal phenacetin (PN) O-deethylation as a CYP1A2 index, microsomal and cytosolic p-nitrophenyl acetate (NPA) hydrolysis as a carboxylesterase index and cytosolic 4-OH-PL sulfation as a sulfotransferase index. The pretreatment of Hep G2 cells with LPZ or BNF increased microsomal ER O-deethylase activities, and the potency of BNF was much higher than that of LPZ. Cytosolic 4-OH-PL sulfation was also elevated by the pretreatment with BNF but not with LPZ. Microsomal PN O-deethylase activity was not detectable in either the control or BNFpretreated group under the conditions used. Microsomal and cytosolic NPA hydrolase activities were similar between the control and the BNF-pretreated groups. Cytotoxicities of PL and 4-OH-PL were attenuated in BNFpretreated Hep G2 cells compared to non-pretreated Hep G2 cells. These results suggest that increased activities of microsomal CYP1A1 and cytosolic sulfotransferases by pretreatment with BNF may contribute to the attenuating the cytotoxicity of PL and 4-OH-PL in Hep G2 cells, at least in part.