Propanediol dehydratase system. Role of monovalent cations in binding of vitamin B12 coenzyme or its analogs to apoenzyme

Abstract

Holopropanediol dehydratase (EC 4.2.1.28) was resolved completely into the apoenzyme and corrinoid(s) upon gel filtration on a column of Sephadex G-25 in the absence of potassium ion. The apoprotein obtained by this resolution procedure could be reconstituted into the catalytically active holoenzyme by the incubation at 37' with added B12 coenzyme in the presence of potassium ion. The recovery of dehydratase activity was nearly quantitative. The major corrinoid recovered was identified as 5 '-deoxyadenosylcobalamin. Of inactive complexes between the apoenzyme and irreversible cobamide inhibitors of propanediol dehydratase, cyanocobalamin-and methylcobalamin-apoenzyme complexes were also mostly resolved upon the gel filtration in the absence of both potassium ion and the substrate, yielding the apoenzyme which was reconstitutable into the active holoenzyme. Hydroxocobalamin-apoenzyme complex, however, was hardly resolvable under the same conditions. In the presence of both potassium ion and the substrate, gel filtration of the holo-I t has been generally considered that vitamin B12 coenzyme or its analogs bind to the apoprotein of propanediol dehydratase (DL-1 ,Zpropanediol hydro-lyase, EC 4.2.1.28) or to those of many other Ble coenzyme-dependent enzymes almost irreversibly and the resolution of B12 coenzyme-apoenzyme complex (holoenzyme) or coenzyme analog-apoenzyme complex into the active apoprotein and corresponding cobalamin is usually accompanied by a significant loss of the enzyme activity. Charcoal treatment or exhaustive dialysis was found not to be effective. An acid ammonium sulfate resolution procedure, which was effectively used for mammalian methylmalonyl-CoA mutase (Lengyel et al., 1960) and ethanolamine deaminase (Kaplan and Stadtman, 1968b), led to a marked decrease in the propanediol dehydratase activity (T. Toraya, S. Shimizu, and S. Fukui, 1969, unpublished results).During the course of an investigation on factors necessary for binding of B12 coenzyme or its analogs to the apoprotein of propanediol dehydratase, our attention was drawn to the function of a monovalent cation which was required as a cofactor by this enzyme. Although it has previouslycbeen reported that propanediol dehydratase and many other coenzyme BIZ dependent enzymes show an absolute requirement for a monovalent cation, such as potassium ion, for their catalytic activitieslSmiley and Sobolov. Receiued March 15, 1971. A preliminary report of portions of this work has been published (Toraya er al., 1970).To whom correspondence should be addressed. enzyme or coenzyme analog-apoenzyme complexes resulted in negligible resolution. When gel filtration of the holoenzyme was carried out in the presence of potassium ion only, the holoenzyme was found to be resolved according to the kinetics of first-order reaction. These experiments indicate that potassium ion plays an essential role in the binding of the apoenzyme with coenzyme B I~ or its analogs except for hydroxocobalamin. Only hydroxocobalamin could bind to the apoenzyme even in t...

Dissimilation of 1,2-propanediol by rumen micro-organisms

“…The most extensive studies of metabolism of I ,a-propanediol, mainly in Aerobacter aerogenes, were made by Abeles and co-workers. It was concluded that the dehydration of I ,a-propanediol to propionaldehyde appears to be an intramolecular oxidation-reduction reaction requiring vitamin BIZ coenzyme (Abeles & Lee, 1961) and that the coenzyme serves as hydrogen carrier (Brownstein & Abeles, 1961) in the dchydratase system that also involves monovalent cations (Toraya, Sugimoto, Tamao, Shimizu & Fukui, 1971). The object of our work was to study the metabolism of 1,2-propanediol in the rumen in greater detail, with particular emphasis on the end-products and upon the involvement of the metabolism of the diol in the economy of metabolic hydrogen in the rumen.…”

Construction and Characterization of Hybrid Dehydratases between Adenosylcobalamin-Dependent Diol and Glycerol Dehydratases

“…Specificity of hybrid enzymes for monovalent cations was determined by measuring the enzyme activity with 1,2-propanediol as substrate in the presence of monovalent cations (12). Figure 4 shows the relationship between ionic radius and relative cofactor activity of each metal ion.…”

“…Large and small components correspond to the ␣ 2 ␥ 2 complex and the ␤ subunit, respectively ( 6, 7 ). Their catalytic properties are similar, but they are different in the binding affinity for AdoCbl ( 8,9 ), substrate specificity ( 1-3, 10, 11 ), susceptibility to suicide inactivation by glycerol ( 2, 9, 10 ), monovalent cation specificity ( 1,11,12 ), and immunochemical reactivity toward anti-diol dehydratase antiserum ( 11 ) (for review, see Refs. 13-15 )).…”

On the cation sensibility of the vitamin B12‐dependent methionine synthetase (5‐methyltetrahydrofolate‐homocysteine‐ methyltransferase from Escherichia coli

“…The molecular mechanism by which activating and inhibiting ions operate cannot be deduced from the experiments described above. It may, however, be supposed that, similarly to the activation of dioldehydratase [6][7][8], the cations act on the enzyme protein conformation thus altering the affinity for B12 or other cofactors and substrates.…”

“…Though these ions only are able to exert relatively weak coordinative forces, the complex formation apparently enables them to keep enzyme proteins in specific biologically active conformations. Some B12-dependent enzymes have turned out to belong to this group of cation-sensitive enzymes [6][7][8][9][10][11][12]. Toraya et al [6][7][8] have shown that the active form of the deoxyadenosyl cobalamin-dependent propanediol dehydratase contains potassium ions which are supposed to facilitate the binding between enzyme and B12 coenzyme thus enhancing the enzymatic activity.…”