Cobalamins Adenosylcobalamin-dependent

Interaction with Adenosylcobalamin. It has been considered generally that adenosylcobalamin or its analogs binds to the apoprotein of diol dehydrase or other adenosylcobalamin-dependent enzymes almost irreversibly (4). However, we found that the holo-enzyme of diol dehydrase was resolved completely into intact apoen-zyme and adenosylcobalamin when subjected to gel filtration on a Sephadex G-25 column in the absence of K+ (9, 10). Among the inactive complexes of diol dehydrase with irreversible cobalamin inhibitors, those with cyanocobalamin and methylcobalamin also were resolved upon gel filtration on Sephadex G-25 in the absence of both K+ and substrate, yielding the apoenzyme, which was reconstitutable into the active holoenzyme (II). The enzyme-hydroxocobalamin complex, however, was not resolvable under the same conditions. The enzyme-cobalamin complexes were not resolved at all by gel filtration in the presence of both K+ and substrate. When gel filtration of the holoenzyme was carried out in the presence of K+ only, the holoen-... 

Although numerous enzymatic reactions requiring vitamin B12 have been described, and 10 reactions for adenosylcobalamin alone have been identified, only three pathways in man have so far been recognized, one of which has only recently been identified (PI). Two of these require the vitamin in the adenosyl form and the other in the methyl form. These cobalamin coenzymes are formed by a complex reaction sequence which results in the formation of a covalent carbon-cobalt bond between the cobalt nucleus of the vitamin and the methyl or 5 -deoxy-5 -adenosyl ligand, with resulting coenzyme specificity. Adenosylcobalamin is required in the conversion of methylmalonate to succinate (Fig. 2), while methylcobalamin is required by a B12-dependent methionine synthetase that enables the methyl group to be transferred from 5-methyltetrahydrofolate to homocysteine to form methionine (Fig. 3). 

S-Methylmalonyl-CoA mutase (EC 5.4.99.2) is a deoxyadenoxyladen-osylcobalamin-dependent enzyme of mitochondria required to catalyze the conversion of methylmalonyl-CoA to succinyl-CoA. A decrease in the activity of methylmalonyl-CoA mutase leads to the urinary excretion of large amounts of methylmalonic acid (C22). The biochemical lesion may be at the mutase level due to an abnormality of apoenzyme protein or an inability to elaborate the required coenzyme form of vitamin B12> i.e., adenosyl-cobalamin. In rare cases the abnormality may be due to an inability to convert the d form of methylmalonyl-CoA mutase to the l form as a result of a defective racemase (EC 5.1.99.1) (Kll). In patients, the nature of the abnormality can be determined by tissue culture studies (D13) and by clinical trial, since patients with a defect in adenosylcobalamin production will show clinical improvement when treated with very large doses of vitamin B12 (Mil). 

Both Components F and S were required for irreversible cleavage of the C-Co bond of adenosylcobalamin by oxygen upon aerobic incubation with the coenzyme in the absence of substrate. This suggests that activation of the C-Co bond of the coenzyme is dependent on both components. Sephadex G-25 filtration experiments showed that neither adenosylcobalamin nor cyanocobalamin was bound by the individual components, F or S. Both of them were necessary for the cobalamin binding (8). 

Numerous analogs of adenosylcobalamin have been tested for their ability to replace or to inhibit the action of the coenzyme in the adenosyl-cobalamin-dependent ribonucleotide reductase reaction the enzyme from L. leichmannii has been used in most of these studies. Kinetic studies have been used in most investigations of analog-enzyme interactions and thus the interpretation of data regarding the affinity of analogs for the reductase is subject to the limitations imposed on kinetic studies of a complex reaction. 

Cobalamins are essential enzymatic cofactors in human biochemistry. Coba-lamins chemical structure is based on the tetrapyrrole ring, while the chemical properties of the Co bond located in the centre of their moiety have been the focus of extensive research. Cyanocobalamin, the most known form of cobalamins, is rarely found in nature. Methylcobalamin and adenosylcobalamin are the two active forms of cobalamins in vivo in humans. Weakening of the Co C bond and its homolytic or heterolytic cleavage have been uncovered as an essential mechanism in the biochemical role of cobalamins as cofactors in humans. Recent studies using modem computational methods and application of quantum chemistry models have widened our knowledge of cobalamins biochemistry and are expected to contribute to our further understanding of cobalamin-dependent enzymes. 

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