Cobalamin-dependent methionine synthase: probing the role of the axial base in catalysis of methyl transfer between methyltetrahydrofolate and exogenous cob(I)alamin or cob(I)inamide

Cobalamin-dependent methionine synthase (MetH) catalyzes the transfer of methyl groups between methyltetrahydrofolate (CH(3)-H(4)folate) and homocysteine, with the enzyme-bound cobalamin serving as an intermediary in the methyl transfers. An MetH fragment comprising residues 2-649 contains modules that bind and activate CH(3)-H(4)folate and homocysteine and catalyze methyl transfers to and from exogenous cobalamin. Comparison of the rates of reaction of cobalamin, which contains a dimethylbenzimidazole nucleotide coordinated to the cobalt in the lower axial position, and cobinamide, which lacks the dimethylbenzimidazole nucleotide, allows assessment of the degree of stabilization the dimethylbenzimidazole base provides for methyl transfer between CH(3)-H(4)folate bound to MetH(2-649) and exogenous cob(I)alamin. When the reactions of cob(I)alamin or cob(I)inamide with CH(3)-H(4)folate are compared, the observed second-order rate constants are 2.7-fold faster for cob(I)alamin; in the reverse direction, methylcobinamide reacts 35-fold faster than methylcobalamin with enzyme-bound tetrahydrofolate. These measurements can be used to estimate the influence of the dimethylbenzimidazole ligand on both the thermodynamics and kinetics of methyl transfer between methyltetrahydrofolate and cob(I)alamin or cob(I)inamide. The free energy change for methyl transfer from CH(3)-H(4)folate to cob(I)alamin is 2.8 kcal more favorable than that for methyl transfer to cob(I)inamide. Dimethylbenzimidazole contributes approximately 0.6 kcal/mol of stabilization for the forward reaction and approximately 2.2 kcal/mol of destabilization for the reverse reaction. Binding of methylcobalamin to full-length methionine synthase is accompanied by ligand substitution, and switching between "base-on" and "base-off" states of the cofactor has been demonstrated [Bandarian, V., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 8156-8163]. The present results disfavor a major role for such switching in catalysis of methyl transfer, and are consistent with the hypothesis that the primary role of the ligand triad in methionine synthase is controlling the distribution of enzyme conformations during catalysis.     

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica serovar typhimurium LT2

Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC 1.6.8.1) as the enzyme responsible for this activity. The fre gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His-tagged Fre reduced flavins (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) and cob(III)alamin to cob(II)alamin very efficiently. Photochemically reduced FMN substituted for Fre in the reduction of cob(III)alamin to cob(II)alamin, indicating that the observed cobalamin reduction activity was not Fre dependent but FMNH(2) dependent. Enzyme-independent reduction of cob(III)alamin to cob(II)alamin by FMNH(2) occurred at a rate too fast to be measured. The thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin was detectable by alkylation of the cob(I)alamin nucleophile with iodoacetate. Detection of the product, caboxymethylcob(III)alamin, depended on the presence of FMNH(2) in the reaction mixture. FMNH(2) failed to substitute for potassium borohydride in in vitro assays for corrinoid adenosylation catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme, even under conditions where Fre and NADH were present in the reaction mixture to ensure that FMN was always reduced. These results were interpreted to mean that Fre was not responsible for the generation of cob(I)alamin in vivo. Consistent with this idea, a fre mutant displayed wild-type cobalamin biosynthetic phenotypes. It is proposed that S. enterica serovar Typhimurium LT2 may not have a cob(III)alamin reductase enzyme and that, in vivo, nonadenosylated cobalamin and other corrinoids are maintained as co(II)rrinoids by reduced flavin nucleotides generated by Fre and other flavin oxidoreductases.     

Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri.

The ultraviolet/visible spectrum of the pure pink-orange 2-methyleneglutarate mutase from Clostridium barkeri between 300-600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen-stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively. Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 +/- 0.3 mol/mol tetrameric enzyme (m = 300 kDa). Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40-50% of the initially bound cobalamin. This preparation could be reactivated to 95-100% by addition of adenosylcobalamin. The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity. 2-Methyleneglutarate mutase was inactivated by incubation with aquo-, cyano- or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin. Upon incubation of the mutase with [5'-3H]adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium-labelled cofactor without loss of activity. During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin. EPR spectroscopy demonstrated the presence of 0.8 mol base-on cob(II)alamin/mol enzyme. Upon addition of 2-methyleneglutarate a second EPR signal of about equal intensity at g = 2.13 arose. The question of whether the oxygen-stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation.     

Binding of Cob(II)alamin to the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii. Identification of dimethylbenzimidazole as the axial ligand

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5'-triphosphates to 2'-deoxynucleoside 5'-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2'-deoxy-2'-methylenecytidine 5'-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.     

Ethanolamine ammonia-lyase has a "base-on" binding mode for coenzyme B(12).

Ethanolamine ammonia-lyase (EAL, EC 4.3.1.7) catalyzes a coenzyme B(12)-dependent deamination of vicinal amino alcohols. The mode of binding of coenzyme B(12) to EAL has been investigated by electron paramagnetic resonance spectroscopy (EPR) using [(15)N]-dimethylbenzimidazole-coenzyme B(12). EAL was incubated with either unlabeled or (15)N-enriched coenzyme B(12) and then either exposed to light or treated with ethanol to generate the cleaved form of the cofactor, cob(II)alamin (B(12r)) bound in the active site. The reaction mixtures were examined by EPR spectroscopy at 77 K. (15)N superhyperfine splitting in the EPR signals of the low-spin Co(2+) of B(12r), bound in the active site of EAL, indicates that the dimethylbenzimidazole moiety of the cofactor contributes the lower axial ligand consistent with "base-on" binding of coenzyme B(12) to EAL.     

Analysis of the Cob(II)alamin-5'-deoxy-3',4'-anhydroadenosyl radical triplet spin system in the active site of diol dehydrase

A triplet spin system (S=1) is detected by low-temperature electron paramagnetic resonance (EPR) spectroscopy in samples of diol dehydrase and the functional adenosylcobalamin (AdoCbl) analogue 5'-deoxy-3',4'-anhydroadenosylcobalamin (anAdoCbl). Different spectra are observed in the presence and absence of the substrate (R,S)-1,2-propanediol. In both cases, the spectra include a prominent half-field transition (DeltaM(S) = 2) that is a hallmark of strongly coupled triplet spin systems. The appearance of 59Co hyperfine splitting in the EPR signals and the positions (g values) of the signals in the spectra show that half of the triplet spin is contributed by the low-spin Co2+ of cob(II)alamin. Line width effects from isotopic labeling (13C and 2H) in the 5'-deoxy-3',4'-anhydroribosyl ring demonstrate that the other half of the spin triplet is from an allylic 5'-deoxy-3',4'-anhydroadenosyl (anhydroadenosyl) radical. The zero-field splitting (ZFS) tensors describing the magnetic dipole-dipole interactions of the component spins of the triplets have rhombic symmetry because of electron spin delocalization within the organic radical component and the proximity of the radical to the low-spin Co2+. The dipole-dipole interaction was modeled as a summation of point-dipole interactions involving the spin-bearing orbitals of the anhydroadenosyl radical and cob(II)alamin. Geometries which are consistent with the ZFS tensors in the presence and absence of the substrate position the 5'-carbon of the anhydroadenosyl radical 3.5 and 4.1 A from Co2+, respectively. Homolytic cleavage of the cobalt-carbon bond of the analogue in the absence of the substrate indicates that, in diol dehydrase, binding of the coenzyme to the protein weakens the bond prior to binding of the substrate.     

Analysis of the electron paramagnetic resonance spectrum of a radical intermediate in the coenzyme B(12)-dependent ethanolamine ammonia-lyase catalyzed reaction of S-2-aminopropanol

The structure of the steady-state radical intermediate in the deamination of S-2-aminopropanol catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been probed by electron paramagnetic resonance (EPR) spectroscopy using isotopically labeled forms of the substrate and of the adenosylcobalamin cofactor. Electron spin-spin coupling between the radical, centered on the carbon skeleton of the substrate, and the low-spin Co(2+) in cob(II)alamin (B(12r)) produces a dominant splitting of the EPR signals of both the radical and the Co(2+). Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by approximately 11 A. Experiments with (13)C- and with (2)H-labeled forms of S-2-aminopropanol show that the radical is centered on C1 of the carbon skeleton of the substrate in agreement with an earlier report [Babior, B. M., Moss, T. H., Orme-Johnson, W. H., and Beinert, H., (1974) J. Biol. Chem. 249, 4537-4544]. Experiments with perdeutero-S-2-aminopropanol and [2-(15)N]-perdeutero-S-2-aminopropanol reveal a strong hyperfine splitting from the substrate nitrogen, which indicates that the radical is the initial substrate radical created by abstraction of a hydrogen atom from C1 of S-2-aminopropanol. The strong nitrogen hyperfine splitting further indicates that the amino substituent at C2 is approximately eclipsed with respect to the half-occupied p orbital at C1. Experiments with adenosylcobalamin enriched in (15)N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co(2+) in a functional steady-state reaction intermediate.     

Mechanism of reductive activation of cobalamin-dependent methionine synthase: an electron paramagnetic resonance spectroelectrochemical study

The mechanism of reductive methylation of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate:homocysteine methyltransferase, EC 2.1.1.13) has been investigated by electron paramagnetic resonance (EPR) spectroelectrochemistry. The enzyme as isolated is inactive, and its UV/visible absorbance and EPR spectra are characteristic of cob(II)alamin. There is an absolute requirement for catalytic amounts of AdoMet and a reducing system for the formation and maintenance of active enzyme during in vitro turnover. The midpoint potentials of the enzyme-bound cob(II)alamin/cob(I)alamin and cob(III)alamin/cob(II)alamin couples have been determined to be -526 +/- 5 and +273 +/- 4 mV (versus the standard hydrogen electrode), respectively. The presence of either CH3-H4folate or AdoMet shifts the equilibrium distribution of cobalamin species observed during reduction by converting cob(I)alamin to methylcobalamin. The magnitude of these shifts is however vastly different, with AdoMet lowering the concentration of cob(II)alamin at equilibrium by a factor of at least 3 X 10(7), while CH3-H4folate lowers it by a factor of 19. These studies of coupled reduction/methylation reactions elucidate the absolute requirement for AdoMet in the in vitro assay system, in which the ambient potential is approximately -350 mV versus the standard hydrogen electrode. At this potential, the equilibrium distribution of cobalamin in the presence of CH3-H4folate would be greatly in favor of the cob(II)alamin species, whereas in the presence of AdoMet the equilibrium favors methylated enzyme. In these studies, a base-on form of cob(II)alamin in which the dimethylbenzimidazole substituent of the corrin ring is the lower axial ligand for the cobalt has been observed for the first time on methionine synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron transfer in the substrate-dependent suicide inactivation of lysine 5,6-aminomutase

The lysine 5,6-aminomutase (5,6-LAM) purified from Clostridium sticklandii was found to undergo rapid inactivation in the absence of the activating enzyme E(2) and ATP. In the presence of substrate, inactivation was also seen for the recombinant 5,6-LAM. This adenosylcobalamin-dependent enzyme is postulated to generate cob(II)alamin and the 5'-deoxyadenosyl radical through enzyme-induced homolytic scission of the Co-C bond. However, the products cob(III)alamin and 5'-deoxyadenosine were observed upon inactivation of 5,6-LAM. Cob(III)alamin production, as monitored by the increase in A(358), proceeds at the same rate as the loss of enzyme activity, suggesting that the activity loss is related to the adventitious generation of cob(III)alamin during enzymatic turnover. The cleavage of adenosylcobalamin to cob(III)alamin is accompanied by the formation of 5'-deoxyadenosine at the same rate, and the generation of cob(III)alamin proceeds at the same rate both aerobically and anaerobically. Suicide inactivation requires the presence of substrate, adenosylcobalamin, and PLP. We have ruled out the involvement of either the putative 5'-deoxyadenosyl radical or dioxygen in suicide inactivation. We have shown that one or more reaction intermediates derived from the substrate or/and the product, presumably a radical, participate in suicide inactivation of 5,6-LAM through electron transfer from cob(II)alamin. Moreover, L-lysine is found to be a slowly reacting substrate, and it induces inactivation at a rate similar to that of D-lysine. The alternative substrate beta-lysine induces inactivation at least 25 times faster than DL-lysine. The inactivation mechanism is compatible with the radical isomerization mechanism proposed to explain the action of 5,6-LAM.     

Reaction of alkylcobalamins with thiols

Carbon-13 NMR spectroscopy and phosphorus-31 NMR spectroscopy have been used to study the reaction of several alkylcobalamins with 2-mercaptoethanol. At alkaline pH, when the thiol is deprotonated, the alkyl-transfer reactions involve a nucleophilic attack of the thiolate anion on the Co-methylene carbon of the cobalamins, yielding alkyl thioethers and cob(II)alamin. In these nucleophilic displacement reactions cob(I)alamin is presumably formed as an intermediate. The higher alkylcobalamins react more slowly than methylcobalamin. The lower reactivity of ethyl- and propylcobalamin is probably the basis of the inhibition of the corrinoid-dependent methyl-transfer systems by propyl iodide. The transfer of the upper nucleoside ligand of adenosylcobalamin to 2-mercaptoethanol is a very slow process; S-adenosyl-mercaptoethanol and cob(II)alamin are the final products of the reaction. The dealkylation of (carboxymethyl)cobalamin is a much more facile reaction. At alkaline pH S-(carboxymethyl)mercaptoethanol and cob(II)alamin are produced, while at pH values below 8 the carbon-cobalt bond is cleaved reductively to acetate and cob(II)alamin. The reductive cleavage of the carbon-cobalt bond of (carboxymethyl)cobalamin by 2-mercaptoethanol is extremely fast when the cobalamin is in the "base-off" form. Because we have been unable to detect trans coordination of 2-mercaptoethanol, we favor a mechanism that involves a hydride attack on the Co-methylene carbon of (carboxymethyl)cobalamin rather than a trans attack of the thiol on the cobalt atom.     

Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase

The final step in the conversion of vitamin B(12) into coenzyme B(12) (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransferase (ATR). Prior studies identified the human ATR and showed that defects in its encoding gene underlie cblB methylmalonic aciduria. Here two common polymorphic variants of the ATR that are found in normal individuals are expressed in Escherichia coli, purified, and partially characterized. The specific activities of ATR variants 239K and 239M were 220 and 190 nmol min(-1) mg(-1), and their K(m) values were 6.3 and 6.9 mum for ATP and 1.2 and 1.6 mum for cob(I)alamin, respectively. These values are similar to those obtained for previously studied bacterial ATRs indicating that both human variants have sufficient activity to mediate AdoCbl synthesis in vivo. Investigations also showed that purified recombinant human methionine synthase reductase (MSR) in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin that was adenosylated to AdoCbl by ATR. The optimal stoichiometry for this reaction was approximately 4 MSR/ATR and results indicated that MSR and ATR physically interacted in such a way that the highly reactive reaction intermediate [cob(I)alamin] was sequestered. The finding that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR reduced cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for MSR.     

Identification of cis-ethanesemidione as the organic radical derived from glycolaldehyde in the suicide inactivation of dioldehydrase and of ethanolamine ammonia-lyase

The hydrate of glycolaldehyde is a substrate analogue that induces the formation of cob(II)alamin and 5'-deoxyadenosine from adenosylcobalamin at the active site of dioldehydrase, and the resulting complex is inactive. The carbon atoms of glycolaldehyde hydrate remain bound to this complex, and it has been postulated that the first step or steps of the catalytic process on glycolaldehyde hydrate generate an intermediate that undergoes a destructive side reaction leading to inactivation of the enzyme [Wagner, O. W., Lee, H. A., Jr., Frey, P. A., and Abeles, R. H. (1966) J. Biol. Chem. 249, 1751-1762]. All evidence suggests that dioldehydrase reaction proceeds by a radical mechanism, and the glycolaldehyde hydrate is expected to be converted initially into a radical. Electron paramagnetic resonance (EPR) spectroscopic analysis of the inactivated complex shows that glycolaldehyde is transformed into a cis-ethanesemidione radical that is weakly spin-coupled to the cob(II)alamin in the active site of the enzyme. This radical has been identified by analysis of EPR spectra obtained from samples with (13)C- and (2)H-labeled forms of glycolaldehyde. The analysis shows that the stable radical associated with the inactive complex is symmetrical and that it contains a single solvent-exchangeable proton, consistent with a cis-ethanesemidione. Glycolaldehyde also inactivates ethanolamine ammonia-lyase (EAL). EPR studies of ethanolamine ammonia-lyase reveal that treatment with glycolaldehyde also results in formation of an ethanesemidione radical bound in the active site. The suicide inactivation in both enzymatic reactions is postulated to result from formation of this stable radical, which cannot react further to abstract a hydrogen atom from 5'-deoxyadenosine. Analysis of the electron spin-spin coupling between the semidione radicals and cob(II)alamin in both enzymes indicates that the distance between the radical and Co(2+) is approximately 11 A in each case.     

Hydrazine cation radical in the active site of ethanolamine ammonia-lyase: mechanism-based inactivation by hydroxyethylhydrazine.

A study has been made of the mechanism of inactivation of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase (EAL), by hydroxyethylhydrazine. Incubation of EAL with adenosylcobalamin and hydroxyethylhydrazine, an analogue of ethanolamine, leads to rapid and complete loss of enzymic activity. Equimolar quantities of 5'-deoxyadenosine, cob(II)alamin (B(12r)), hydrazine cation radical, and acetaldehyde are products of the inactivation. Inactivation is attributed to the tight binding of B(12r) in the active site. Removal of B(12r) from the protein by ammonium sulfate precipitation under acidic conditions, however, restores significant activity. This inactivation event has also been monitored by electron paramagnetic resonance (EPR) spectroscopy. In addition to EPR signals associated with B(12r), spectra of samples of inactivation mixtures reveal the presence of another radical. The other radical is bound in the active site where it undergoes weak magnetic interactions with the low spin Co(2+) in B(12r). The radical species was unambiguously identified as a hydrazine cation radical by using [(15)N(2)]hydroxyethylhydrazine, (2)H(2)O, and quantitative interpretation of the EPR spectra. Homolytic fragmentation of a hydroxyethylhydrazine radical to acetaldehyde and a hydrazine cation radical is consistent with all of the observations. All of the experiments indicate that the mechanism-based inactivation of EAL by hydroxyethylhydrazine results from irreversible cleavage of the cofactor and tight binding of B(12r) to the active site.     

Multiple roles of ATP:cob(I)alamin adenosyltransferases in the conversion of B12 to coenzyme B12

Our mechanistic understanding of the conversion of vitamin B₁₂ into coenzyme B₁₂ (a.k.a. adenosylcobalamin, AdoCbl) has been substantially advanced in recent years. Insights into the multiple roles played by ATP:cob(I)alamin adenosyltransferase (ACA) enzymes have emerged through the crystallographic, spectroscopic, biochemical, and mutational analyses of wild-type and variant proteins. ACA enzymes circumvent the thermodynamic barrier posed by the very low redox potential associated with the reduction of cob(II)alamin to cob(I)alamin by generating a unique four-coordinate cob(II)alamin intermediate that is readily converted to cob(I)alamin by physiological reductants. ACA enzymes not only synthesize AdoCbl but also they deliver it to the enzymes that use it, and in some cases, enzymes in which its function is needed to maintain the fidelity of the AdoCbl delivery process have been identified. Advances in our understanding of ACA enzyme function have provided valuable insights into the role of specific residues, and into why substitutions of these residues have profound negative effects on human health. From an applied science standpoint, a better understanding of the adenosylation reaction may lead to more efficient ways of synthesizing AdoCbl.     

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

[Omega-(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 kcat and kcat/Km values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regular coenzyme with diol dehydratase, respectively. The kcat/kinact 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 kcat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonia-lyase, 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.     

9-(Adenylyl)alkylcobalamins as inhibitors of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes

The behavior of two coenzyme analogs, [(5-aden-9-yl)methoxyethyl] cob (III) alamin and [(5-aden-9-yl)pentyl] cob (III) alamin modified at the nucleoside ligand sugar moiety was studied in the system of adenosyl-cobalamin-dependent glycerol dehydratase from Aerobacter aerogenes. It was shown that neither of the analogs possesses coenzyme properties and that both are strong competitive inhibitors for adenosylcobalamin (AdoCbl). The affinity of the two analogs for the apoenzyme is higher than that of AdoCbl. The data obtained are indicative of the essential role of the ribofuranoside fragment of AdoCbl in the manifestation of the coenzyme activity. The apoenzyme interaction with the analogs under study is discussed in terms of the Dreiding stereomodels for AdoCbl and its analogs.     

Functional characterization and mutation analysis of human ATP:Cob(I)alamin adenosyltransferase

ATP:cob(I)alamin adenosyltransferase catalyzes the final step in the conversion of vitamin B 12 into the active coenzyme, adenosylcobalamin. Inherited defects in the gene for the human adenosyltransferase (hATR) result in methylmalonyl aciduria (MMA), a rare but life-threatening illness. In this study, we conducted a random mutagenesis of the hATR coding sequence. An ATR-deficient strain of Salmonella was used as a surrogate host to screen for mutations that impaired hATR activity in vivo. Fifty-seven missense mutations were isolated. These mapped to 30 positions of the hATR, 25 of which had not previously been shown to impair enzyme activity. Kinetic analysis and in vivo tests for enzyme activity were performed on the hATR variants, and mutations were mapped onto a hATR structural model. These studies functionally defined the hATR active site and tentatively implicated three amino acid residues in facilitating the reduction of cob(II)alamin to cob(I)alamin which is a prerequisite to adenosylation.     

Mechanism of Lactobacillus leichmannii ribonucleotide reductase studied with Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (Pseudocoenzyme B12) as coenzyme.

Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (pseudocoenzyme B12) purified from Clostridium tetanomorphum has been reacted with ribonucleotide reductase purified from Lactobacillus leichmannii under various conditions, and the properties of the products obtained have been compared by electron paramagnetic resonance (EPR) with those previously reported for products formed from the normal coenzyme (adenosylcobalamin). The rapidly formed intermediate and the slowly formed "doublet" species from the pseudocoenzyme have EPR spectra identical with those formed from the normal coenzyme. This and other considerations make it less likely that the unusual magnetic properties of the rapidly formed intermediate are due to strongly distorted octahedral symmetry about Co(II) as previously postulated. Instead it is probable that the EPR spectrum is due to interaction of the radical pair by both exchange coupling and magnetic dipole--dipole coupling. Although Coalpha-[alpha-(aden-9-YL)]cob(II)amide in solution does not show superhyperfine splitting in the EPR spectrum because of its base-off configuration, the cob(II)amide formed by degradation of the pseudocoenzyme within the catalytic site of the enzyme did show triplets due to a nitrogen axially coordinated to cobalt. This suggests that binding of the cob(II)amide to the reductase catalytic site causes a shift to the base-on form.