Hydrogen abstraction from thiols by adenosyl radicals: chemical precedent for thiyl radical formation, the first catalytic step in ribonucleoside triphosphate reductase from Lactobacillus leichmannii

Aqueous solutions of adenosylcobalamin (AdoCbl) were thermolyzed with excess beta-mercaptoethanol under anaerobic conditions. The product studies reveal that approximately 90% Co-C bond homolysis occurs, to yield Co(II)cobalamin, 5'-deoxyadenosine, and the disulfide product from the combination of two HOCH2CH2S* radicals, 2,2'-dithiodiethanol; there is also approximately 10% Co-C bond heterolysis, yielding Co(III)cobalamin, adenine, and 2,3-dihydroxy-4-pentenal. The kinetic studies show there is a first-order dependence on AdoCbl and zero-order dependence on thiol under the higher [RSH] experimental conditions used, consistent with the rate-determining step at high [RSH] being the generation of Ado*. The kinetic results require that, in enzyme-free AdoCbl solution, adenosyl radical (Ado*) is formed as a discrete intermediate which then abstracts H* from the added thiol. The activation parameters for Co-C bond homolysis in the presence of thiol trap are the same within experimental error as the activation parameters for Co-C bond homolysis without trap, standard delta H(obs) = 29(2) kcal mol(-1) and standard delta S(obs) = -1(5) e.u. The results, in comparison to the rate of Co-C bond homolysis in ribonucleoside triphosphate reductase (RTPR), reveal that RTPR accelerates Co-C bond cleavage in AdoCbl by approximately 10(10+/-1). The recent literature evidence bearing on the exact mechanism of RTPR enzymic cleavage of the Co-C bond of AdoCbl is briefly discussed, notably the fact that this mechanism is presently controversial, but does involve at least coupled (and possibly concerted) Co-C cleavage, -S-H cleavage, and C-H (Ado-H) formation steps.     

Adenosylcobalamin-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii. Rapid, improved purification involving dGTP-based affinity chromatography plus biophysical characterization studies demonstrating enhanced, "crystallographic level" purity.

Ribonucleoside triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii is a 5'-deoxyadenosylcobalamin-dependent (AdoCbl; Coenzyme B12) enzyme. RTPR is also a prototypical adenosylcobalamin-dependent ribonucleotide reductase, one that, as its name indicates, converts ribonucleoside triphosphates (NTP) to deoxyribonucleoside triphosphates (dNTP). Upon substrate binding to RTPR, AdoCbl's cobalt-carbon bond is cleaved to generate cob(II)alamin, 5'-deoxyadenosine, and the cysteine (C408) derived thiyl radical. Five key cysteines (Cys 119, 408, 419, 731, and 736), from among the ten total cysteines, are involved in RTPR's catalytic mechanism. A critical examination of the RTPR isolation and purification literature suggested that the purification protocol currently used results in RTPR which contains 2040% microheterogeneity, along with minor contamination by other proteins. In addition, no report of crystalline RTPR has ever appeared. The literature indicates that irreversible cysteine oxidation (e.g., to -SO2H or -SO3H) is one highly plausible reason for the microheterogeneity of RTPR. The literature also indicates that improvement in the level of enzyme purity is the most effective next step in coaxing enzymes to crystallize that have previously failed to do so. A shortened, improved purification of RTPR has been developed, one involving a shorter purification time, a lower pH, a higher concentration of the more effective reductant DTT (all designed to help protect the cysteines from oxidation), and a final step utilizing our recently reported, improved dGTP-based affinity chromatography resin. The resultant RTPR is approximately 20-30% higher in both specific activity and in its ability to undergo single turnovers, and is homogeneous by mass spectrometry and dynamic light scattering. Additionally, the revised purification procedure eliminates > 30 proteins present in 2-3% amounts along with damaged RTPR that does not bind properly (i.e. tightly) to the dGTP-affinity resin. Finally, dGTP-based affinity chromatography purified RTPR has yielded the first reported, albeit small, single crystals of RTPR.     

The coenzyme b12 analog 5'-deoxyadenosylcobinamide-gdp supports catalysis by methylmalonyl-coa mutase in the absence of trans-ligand coordination

Methylmalonyl-CoA mutase is an 5'-adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to cobalt in solution, is replaced by His(610) donated by the active site. The role of the lower axial ligand in the trillion-fold labilization of the upper axial cobalt-carbon bond has been the subject of enduring debate in the model inorganic literature. In this study, we have used a cofactor analog, 5'deoxyadenosylcobinamide GDP (AdoCbi-GDP), which reconstitutes the enzyme in a "histidine-off" form and which allows us to evaluate the contribution of the lower axial ligand to catalysis. The k(cat) for the enzyme in the presence of AdoCbi-GDP is reduced by a factor of 4 compared with the native cofactor AdoCbl. The overall deuterium isotope effect in the presence of AdoCbi-GDP ((D)V = 7.2 +/- 0.8) is comparable with that observed in the presence of AdoCbl (5.0 +/- 0.6) and indicates that the hydrogen transfer steps in this reaction are not significantly affected by the change in coordination state of the bound cofactor. These surprising results are in marked contrast to the effects ascribed to the corresponding lower axial histidine ligands in the cobalamin-dependent enzymes glutamate mutase and methionine synthase.     

Interaction of 3'-[3H]2'-Chloro-2'-deoxyuridine 5'-triphosphate with ribonucleotide reductase from Lactobacillus leichmannii

Incubation of [3'-3H]2'-chloro-2'-deoxyuridine 5'-triphosphate (CldUTP) with adenosylcobalamin (AdoCbl), reductant, and ribonucleotide reductase from Lactobacillus leichmannii results in the production of 3H2O, uracil, tripolyphosphate, 5'-deoxyadenosine, and cob(II)alamin. The rate of 3H2O release (0.19 mumol/min/mg) is almost identical with the rate of UTP reduction (0.24 mumol/min/mg). The amount of 3H2O release is dependent upon the enzyme to cofactor ratio. With a ribonucleotide reductase: AdoCbl ratio of 1:1000, approximately 500 eq of 3H2O are released. At this time the enzyme is still active, but further destruction of cofactor and turnover of CldUTP is prevented by competitive inhibition of Co(II) + 5'-deoxyadenosine with AdoCbl for binding to ribonucleotide reductase. The 5'-deoxyadenosine and AdoCbl reisolated during incubation of [3'-3H]CldUTP and ribonucleotide reductase contains no detectable radioactivity.     

Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii: Kinetic Evaluation of a Series of Adenosylcobalamin Competitive Inhibitors, [ω-(Adenosin-5′-O-yl)alkyl]cobalamins, Which Mimic the Post Co-C Homolysis Intermediate

A series of [ω-(adenosin-5′-O-yl)alkyl]cobalamins were examined for their inhibitory properties of ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii in the presence of 5′-deoxyadenosylcobalamin (AdoCbl, Coenzyme B₁₂). These AdoCbl analogs, in which oligomethylene chains (C₃-C₇) were inserted between the corrin Co-atom and a 5′-O-atom of the adenosine moiety, were designed to probe the Co-C bond posthomolysis state in AdoCbl-dependent enzymes, a state in which the Co and 5′-C distance is believed to be significantly increased. Experimentally, all five analogs were competitive inhibitors, with K_i in the range of 8–56 μM. The [ω-(adenosin-5′-O-yl)alkyl]cobalamin analog with C₅ methylene carbons was the strongest inhibitor. This same pattern of inhibition, in which the C₅-analog is the strongest inhibitor, was previously observed in the AdoCbl-dependent eliminase enzyme systems, diol dehydratase and glycerol dehydratase. However, in methylmalonyl CoA mutase, the strongest inhibition is by the C₆-analog. This supports the hypothesis that the cobalamin posthomolysis intermediate in the eliminase enzymes differs from that in the mutase enzymes. These findings led, in turn, to an examination of the visible spectra of enzyme-bound cob(II)alamin in these two subclasses of AdoCbl-dependent enzymes. The results reveal an additional insight into the difference between the two classes: in the eliminases, the γ-band of bound cob(II)alamin is shifted from the 473 nm for free cob(II)alamin to longer wavelengths, 475–480 nm. However, in mutases, the γ-band of bound cob(II)alamin is shifted to shorter wavelengths, 465–470 nm. Overall, the results (a) provide strong evidence that two subclasses of AdoCbl-dependent enzymes exist, (b) give insights into the probable posthomolysis state in RTPR and other eliminases, and (c) identifies the C₅-analog as the tightest-binding analog for crystallization and other biophysical studies.     

Solution structure, enzymatic, and non-enzymatic reactivity of 3-isoadenosylcobalamin, a structural isomer of coenzyme B12 with surprising coenzymic activity

The coenzymic activity of eight analogs of coenzyme B(12) (5'-deoxyadenosyl-cobalamin, AdoCbl) with structural alterations in the Ado ligand has been investigated with the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. Six of the analogs were partially active coenzymes, and one, 3-iso-5'-deoxyadenosylcobalamin (3-IsoAdoCbl) was nearly as active as AdoCbl itself. NMR-restrained molecular modeling of 3-IsoAdoCbl revealed a highly conformationally mobile structure which required a four state model to be consistent with the NMR data. Thus, two conformations, one with the IsoAdo ligand over the eastern quadrant of the corrin, and one with the IsoAdo ligand over the northern quadrant, each undergo a facile syn/anti conformational equilibrium in the IsoAdo ligand. Spectrophotometric measurement of the kinetics of RTPR-induced cleavage of the carbon-cobalt bond of 3-IsoAdoCbl showed that it binds to the enzyme with the same affinity as AdoCbl, but its homolysis is only 20% as rapid. Investigation of the non-enzymatic thermolysis of 3-IsoAdoCbl showed that like AdoCbl, 3-IsoAdoCbl decomposes by competing homolytic and heterolytic pathways. A complete temperature-dependent kinetic and product analysis, followed by correction for the base-off species permitted deconvolution of the specific rate constant for both pathways. Eyring plots for the homolysis and heterolysis rate constant cross at 93 degrees C, so that homolysis is the predominant pathway at high temperature, but heterolysis is the predominant pathway at low temperature. At 37 degrees C, the homolysis of 3-IsoAdoCbl is 5.5-fold faster than that of AdoCbl, and the enzyme catalyzes carbon-cobalt bond homolysis in 3-IsoAdoCbl by a factor of 5.9 x 10(7), only 3.9% of the catalytic efficiency with AdoCbl itself. It seems likely that the conformational flexibility of 3-IsoAdoCbl allows it to adopt a coformation in which the hydrogen bonding patterns of the adenine moiety are similar to those of AdoCbl itself, and that this is responsible for the high enzymatic activity of this analog.     

Importance of the histidine ligand to coenzyme B12 in the reaction catalyzed by methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is an adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to the cobalt in solution, is replaced by His-610 donated by the active site. The contribution of the lower axial base to the approximately 10(12)-fold rate acceleration of the homolytic cleavage of the upper axial cobalt-carbon bond has been the subject of intense scrutiny in the model inorganic literature. In contrast, trans ligand effects in methylmalonyl-CoA mutase and indeed the significance of the ligand replacement are poorly understood. In this study, we have used site-directed mutagenesis to create the H610A and H610N variants of methylmalonyl-CoA mutase and report that both mutations exhibit both diminished activity (5,000- and 40,000-fold, respectively) and profoundly weakened affinity for the native cofactor, AdoCbl. In contrast, binding of the truncated cofactor analog, adenosylcobinamide, lacking the nucleotide tail, is less impaired. The catalytic failure of the His-610 mutants is in marked contrast to the phenotype of the adenosylcobinamide-GDP reconstituted wild type enzyme that exhibits only a 4-fold decrease in activity, although His-610 fails to coordinate when this cofactor analog is bound. Together, these studies suggest that His-610 may: (i) play a structural role in organizing a high affinity cofactor binding site possibly via electrostatic interactions with Asp-608 and Lys-604, as suggested by the crystal structure and (ii) play a role in catalyzing the displacement of dimethylbenzimidazole thereby facilitating the conformational change that must precede cofactor docking to the mutase active site.     

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence that the hydrogen transfer mechanism involves a second intermediate hydrogen carrier in addition to the cofactor

Ethanolamine ammonia-lyase catalyzes the adenosylcobalamin (AdoCbl)-dependent conversion of ethanolamine to acetaldehyde and ammonia. During this reaction, a hydrogen atom migrates from the carbinol carbon of ethanolamine to the methyl carbon of acetaldehyde. Previous studies have shown that this migrating hydrogen equilibrates with the hydrogens on the 5'-(cobalt-linked) carbon of the cofactor. On the basis of those studies, a two-step mechanism for hydrogen transfer has been postulated in which the migrating hydrogen is first transferred from the substrate to the cofactor, then in a subsequent step is returned from the cofactor to the product. We now show that this migrating hydrogen is transferred not only to the cofactor, but also to a second acceptor at the active site. Hydrogens on this acceptor do not exchange with water during the course of the reaction, but are released to water when the enzyme is denatured. The catalytic significance of this second hydrogen acceptor was demonstrated by the findings that the transfer of hydrogen to this acceptor required both AdoCbl and active enzyme and that hydrogen at the second acceptor site could be washed out by unlabeled ethanolamine. On the basis of these results, we propose an expanded hydrogen transfer mechanism in which AdoCbl and the second acceptor site serve as alternative intermediate hydrogen carriers during the course of ethanolamine deamination.     

5'-Deoxyadenosine contacts the substrate radical intermediate in the active site of ethanolamine ammonia-lyase: 2H and 13C electron nuclear double resonance studies

The mechanism of propagation of the radical center between the cofactor, substrate, and product in the adenosylcobalamin- (AdoCbl) dependent reaction of ethanolamine ammonia-lyase has been probed by pulsed electron nuclear double resonance (ENDOR) spectroscopy. The radical of S-2-aminopropanol, which appears in the steady state of the reaction, was used in ENDOR experiments to determine the nuclear spin transition frequencies of (2)H introduced from either deuterated substrate or deuterated coenzyme and of (13)C introduced into the ribosyl moiety of AdoCbl. A (2)H doublet (1.4 MHz splitting) was observed centered about the Larmor frequency of (2)H. Identical ENDOR frequencies were observed for (2)H irrespective of its mode of introduction into the complex. A (13)C doublet ENDOR signal was observed from samples prepared with [U-(13)C-ribosyl]-AdoCbl. The (13)C coupling tensor obtained from the ENDOR powder pattern shows that the (13)C has scalar as well as dipole-dipole coupling to the unpaired electron located at C1 of S-2-aminopropanol. The dipole-dipole coupling is consistent with a distance of 3.4+/-0.2 A between C1 of the radical and C5' of the labeled cofactor component. These results establish that the C5' carbon of the 5'-deoxyadenosyl radical moves approximately 7 A from its position as part of AdoCbl to a position where it is in contact with C1 of the substrate which lies approximately 12 A from the Co(2+) of cob(II)alamin. These findings are also consistent with the contention that 5'-deoxyadenosine is the sole mediator of hydrogen transfers in ethanolamine ammonia-lyase.     

Epimerization at carbon-5' of (5'R)-[5'-2H]adenosylcobalamin by ribonucleoside triphosphate reductase: cysteine 408-independent cleavage of the Co-C5' bond

The adenosylcobalamin-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of ribonucleoside triphosphates to deoxyribonucleoside triphosphates. RTPR also catalyzes the exchange of the C5'-hydrogens of adenosylcobalalamin with solvent hydrogen. A thiyl radical located on Cys 408 is generated by reaction of adenosylcobalamin at the active site and is proposed to be the intermediate for both the nucleotide reduction and the 5'-hydrogen exchange reactions. In the present research, a stereochemical approach is used to study the mechanism of the Co-C5' bond cleavage of adenosylcobalamin in the reaction of RTPR. When stereoselectively deuterated coenzyme, (5'R)-[5'-(2)H(1)] adenosylcobalamin (5'R/S = 3:1), was incubated with RTPR or the Cys 408 viariants, C408A-RTPR and C408S-RTPR in the presence of dGTP, the deuterium at the 5'-carbon was stereochemically scrambled, leading to epimerization of the (5'S)-[5'-(2)H(1)]- and (5'R)-[5'-(2)H(1)]-isotopomers. Observation of epimerization with mutated RTPR proves that transient cleavage of the Co-C5' bond occurs in the absence of the thiol group on Cys 408. The rate constants for epimerization by RTPR, C408A-RTPR, and C408S-RTPRs in the presence of dGTP are 5.1, 0.28, and 0.42 s(-1), respectively. Only the wild-type RTPR catalyzes the 5'-hydrogen exchange reaction. Both epimerization and 5'-hydrogen exchange reactions are stimulated by the allosteric effector dGTP, and epimerization is not detected in the absence of the effector. Mechanistic implications with respect to wt-RTPR-mediated carbon cobalt bond homolysis and the intermediacy of the 5'-deoxyadenosyl radical will be presented.     

Assembly and protection of the radical enzyme, methylmalonyl-CoA mutase, by its chaperone

MeaB is a recently described P-loop GTPase that plays an auxiliary role in the reaction catalyzed by the radical B12 enzyme, methylmalonyl-CoA mutase. Defects in the human homologue of MeaB result in methylmalonic aciduria, but the role of this protein in coenzyme B12 assimilation and/or utilization is not known. Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA that uses reactive radical intermediates that are susceptible to oxidative inactivation. In this study, we have examined the influence of MeaB on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase and on the thermodynamics of cofactor binding. MeaB alone has a modest effect on the affinity of the mutase for the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor, increasing it 2-fold from 404 +/- 71 to 210 +/- 22 nM. However, in the presence of GDP, the affinity for the cofactor decreases 5-fold to 1.89 +/- 0.33 microM, while in the presence of guanosine 5'(beta-gamma imino)triphosphate, a nonhydrolyzable analogue of GTP, the binding of AdoCbl to the mutase is not detected. Protection against oxidative inactivation of the mutase by MeaB is dependent upon the presence of nucleotides with the MeaB/GDP and MeaB/GTP complexes decelerating the rate of formation of oxidized cofactor by 3- and 15-fold, respectively. This study suggests that MeaB functions in the GTP-dependent assembly of holomethylmalonyl-CoA mutase and subsequent protection of radical intermediates during catalysis.     

Role of the dimethylbenzimidazole tail in the reaction catalyzed by coenzyme B12-dependent methylmalonyl-CoA mutase

The recent structures of cobalamin-dependent methionine synthase and methylmalonyl-CoA mutase have revealed a striking conformational change that accompanies cofactor binding to these proteins. Alkylcobalamins have octahedral geometry in solution at physiological pH, and the lower axial coordination position is occupied by the nucleotide, dimethylbenzimidazole ribose phosphate, that is attached to one of the pyrrole rings of the corrin macrocycle via an aminopropanol moiety. In contrast, in the active sites of these two B12-dependent enzymes, the nucleotide tail is held in an extended conformation in which the base is far removed from the cobalt in cobalamin. Instead, a histidine residue donated by the protein replaces the displaced intramolecular base. This unexpected mode of cofactor binding in a subgroup of B12-dependent enzymes has raised the question of what role the nucleotide loop plays in cofactor binding and catalysis. To address this question, we have synthesized and characterized two truncated cofactor analogues: adenosylcobinamide and adenosylcobinamide phosphate methyl ester, lacking the nucleotide and nucleoside moieties, respectively. Our studies reveal that the nucleotide tail has a modest effect on the strength of cofactor binding, contributing approximately 1 kcal/mol to binding. In contrast, the nucleotide has a profound influence on organizing the active site for catalysis, as evidenced by the retention of the base-off conformation in the truncated cofactor analogues bound to the mutase and by their inability to support catalysis. Characterization of the kinetics of adenosylcobalamin (AdoCbl) binding by stopped-flow fluorescence spectroscopy reveals a pH-sensitive step that titrates to a pKa of 7.32 +/- 0.19 that is significantly different from the pKa of 3.7 for dimethylbenzimidazole in free AdoCbl. In contrast, the truncated cofactors associate very rapidly with the enzyme at rates that are too fast to measure. Based on these observations, we propose a model in which the base-on to base-off conformational change is slow and is assisted by the enzyme, and is followed by a rapid docking of the cofactor in the active site.     

Riboswitch effectors as protein enzyme cofactors

The recently identified glmS ribozyme revealed that RNA enzymes, like protein enzymes, are capable of using small molecules as catalytic cofactors to promote chemical reactions. Flavin mononucleotide (FMN), S-adenosyl methionine (SAM), adenosyl cobalamin (AdoCbl), and thiamine pyrophosphate (TPP) are known ligands for RNA riboswitches in the control of gene expression, but are also catalytically powerful and ubiquitous cofactors in protein enzymes. If RNA, instead of just binding these molecules, could harness the chemical potential of the cofactor, it would significantly expand the enzymatic repertoire of ribozymes. Here we review the chemistry of AdoCbl, SAM, FMN, and TPP in protein enzymology and speculate on how these cofactors might have been used by ribozymes in the prebiotic RNA World or may still find application in modern biology.     

Loss of allostery and coenzyme B12 delivery by a pathogenic mutation in adenosyltransferase

ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein: an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B(12)), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of 1 equiv of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while k(cat) and K(M)(Cob(I)) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease.     

Sequence-structure-function analysis of the bifunctional enzyme MnmC that catalyses the last two steps in the biosynthesis of hypermodified nucleoside mnm5s2U in tRNA

MnmC catalyses the last two steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA. Previously, we reported that this bifunctional enzyme is encoded by the yfcK open reading frame in the Escherichia coli K12 genome. However, the mechanism of its activity, in particular the potential structural and functional dependence of the domains responsible for catalyzing the two modification reactions, remains unknown. With the aid of the protein fold-recognition method, we constructed a structural model of MnmC in complex with the ligands and target nucleosides and studied the role of individual amino acids and entire domains by site-directed and deletion mutagenesis, respectively. We found out that the N-terminal domain contains residues responsible for binding of the S-adenosylmethionine cofactor and catalyzing the methylation of nm(5)s(2)U to form mnm(5)s(2)U, while the C-terminal domain contains residues responsible for binding of the FAD cofactor. Further, point mutants with compromised activity of either domain can complement each other to restore a fully functional enzyme. Thus, in the conserved fusion protein MnmC, the individual domains retain independence as enzymes. Interestingly, the N-terminal domain is capable of independent folding, while the isolated C-terminal domain is incapable of folding on its own, a situation similar to the one reported recently for the rRNA modification enzyme RsmC.     

Interactions of methylmalonyl CoA mutase from normal human fibroblasts with adenosylcobalamin and methylmalonyl CoA: evidence for non-equivalent active sites

We have examined interactions between human methylmalonyl CoA mutase and two critical ligands, its cofactor adenosylcobalamin (AdoCbl) and its substrate methylmalonyl CoA, by performing in vitro experiments with preparations of mutase apoenzyme and holoenzyme from normal cultured human fibroblasts. When extracts are prepared from cells grown in medium containing high concentrations of hydroxocobalamin, a precursor of AdoCbl, mutase activity measured in Tris-containing buffers in the absence of added AdoCbl accounts maximally for only 50% of that activity measured in the presence of excess AdoCbl. A similar result is observed when mutase holoenzyme is formed in vitro by incubating cell extracts containing apoenzyme with AdoCbl and removing excess AdoCbl by gel filtration. When such holoenzyme preparations are heated at 45 °C and then assayed for activity, their thermostability is less than that of mutase holoenzyme heated in the presence of excess cofactor, but far greater than that of mutase apoenzyme. Methylmalonyl CoA modulates these enzyme-coenzyme interactions, since mutase holoenzyme formed in Triscontaining buffers is resolved to apoenzyme upon exposure to substrate. Qualitatively different data are obtained when buffers containing cations other than Tris are used. Under these conditions, mutase activity measured in the absence of added AdoCbl accounts for nearly 100% of the activity measured in the presence of excess cofactor, whether holoenzyme is formed in intact cells in culture or in cell extracts in vitro. Furthermore, holoenzyme formed in vitro in potassium phosphate buffer is not resolved to apoenzyme upon exposure to substrate. We suggest that the “holoenzyme” form of mutase obtained and assayed in Tris-containing buffers is that molecular species with only one of its two potential AdoCbl binding sites occupied in a catalytically active fashion, and that other ions can influence markedly the interactions between mutase, AdoCbl, and methylmalonyl CoA. These data are consistent, therefore, with the hypothesis that the dimeric mutase apoenzyme is characterized, under certain conditions, by nonequivalent active sites.     

Thermolysis of coenzymes B12 at physiological temperatures: activation parameters for cobalt-carbon bond homolysis and a quantitative analysis of the perturbation of the homolysis equilibrium by the ribonucleoside triphosphate reductase from Lactobacillus leichmannii

The kinetics of the thermolysis of 5'-deoxyadenosylcobalamin (AdoCbl, coenzyme B12) in aqueous solution, pH 7.5, have been studied in the temperature range 30-85 degrees C using AdoCbl tritiated at the adenine C2 position and the method of initial rates. Combined with a careful analysis of the distribution of adenine-containing products, the results permit the dissection of the competing rate constants for carbon-cobalt bond homolysis and heterolysis. After correction for the temperature-dependent occurrence of the much less reactive base-off species of AdoCbl, the activation parameters for homolysis of the base-on species were found to be delta H++homo,on = 33.8 +/- 0.2 kcal mol-1 and delta S++homo,on = 13.5 +/- 0.7 cal mol-1 K-1, values not significantly different from those determined by Hay and Finke (J. Am. Chem. Soc. 108 (1986) 4820), in the temperature range 85-115 degrees C. In contrast, the heterolysis of base-on AdoCbl was characterized by a much smaller enthalpy of activation (delta H++het,on = 18.5 +/- 0.2 kcal mol-1) and a negative entropy of activation (delta S++het,on = -34.0 +/- 0.7 cal mol-1 K-1) so that heterolysis, which is minor pathway at elevated temperatures, is the dominant pathway for AdoCbl decomposition at physiological temperatures. Using literature values for the rate constant for the reverse reaction, the equilibrium constant for AdoCbl homolysis at 37 degrees C was calculated to be 7.9 x 10(-18). Comparison with the equilibrium constant for this homolysis at the active site of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii shows that the enzymes shifts the equilibrium constant towards homolysis products by a factor of 2.9 x 10(12) (17.7 kcal mol-1) by binding the thermolysis products with an equilibrium constant of 7.1 x 10(16) M-2, compared to the bonding constant for AdoCbl of 2.4 x 10(4) M-1.     

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Reaction of the enzyme.cofactor complex with 2-aminoacetaldehyde

Ethanolamine ammonia-lyase (EC 4.3.1.7) catalyzes the adenosylcobalamin-dependent deamination of ethanolamine and 2-aminopropanol. Incubation of the enzyme.cofactor complex with 2-aminoacetaldehyde leads to rapid cleavage of the carbon--cobalt bond accompanied by the destruction of the corrinoid portion of the cofactor. During this reaction the adenosyl portion of the cofactor is oxidized to 4',5'-anhydroadenosine, and the aminoacetaldehyde is converted to acetic acid, which remains associated with the enzyme as a noncovalent complex which survives gel filtration. There is no evidence for the alkylation of the corrin metal by the substrate analog. The enzyme.AdoCbl complex is thus able to eliminate an amino group from a substrate analog without the formation of a new alkyl cobalamin in which the analog is a ligand. These observations do not support the participation of what might be termed "substratylcobalamin" as an intermediate in the ammonia migration occurring in reactions catalyzed by ethanolamine ammonia-lyase.     

The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning

Mutations in cobalamin or B₁₂ trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B₁₂ processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ∼116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.     

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.