Characterization of a succinyl-CoA radical-cob(II)alamin spin triplet intermediate in the reaction catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase

The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co(2+) in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co(2+). Labeling of the substrate with (13)C and (2)H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2'-(13)C]methylmalonyl-CoA and [2-(13)C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon alpha to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme.     

Interactions of diol dehydrase and 3',4'-anhydroadenosylcobalamin: suicide inactivation by electron transfer

3',4'-Anhydroadenosylcobalamin (anAdoCbl) is an analogue of the adenosylcobalamin (AdoCbl) coenzyme (Magnusson, O.Th., and Frey, P. A. (2000) J. Am. Chem. Soc. 122, 8807-8813). This compound supports activity for diol dehydrase at 0.02% of that observed with AdoCbl. In a side reaction, however, anAdoCbl induces suicide inactivation by an electron-transfer mechanism. Homolytic cleavage of the Co-C bond of anAdoCbl at the active site of diol dehydrase was observed by spectrophotometric detection of cob(II)alamin. Anaerobic conversion of enzyme bound cob(II)alamin to cob(III)alamin, both in the absence and presence of substrate, indicates that the coenzyme derived 5'-deoxy-3',4'-anhydroadenosine-5'-yl serves as the oxidizing agent. This hypothesis is supported by the stoichiometric formation of 3',5'-dideoxyadenosine-4',5'-ene as the nucleoside cleavage product, as determined by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Experiments performed in deuterium oxide show that a single solvent exchangeable proton is incorporated into the product. These data are consistent with the intermediate formation of a transient allylic anion formed after one electron transfer from cob(II)alamin to the allylic 5'-deoxy-3',4'-anhydroadenosyl radical. Selective protonation at C3' was demonstrated by spectroscopic characterization of the purified product. This study provides an example of suicide inactivation of a radical enzyme brought about by a side reaction of an analogue of the radical intermediate.     

Identification of the 1,2-propanediol-1-yl radical as an intermediate in adenosylcobalamin-dependent diol dehydratase reaction

The reaction catalyzed by adenosylcobalamin-dependent diol dehydratase proceeds by a radical mechanism. A radical pair consisting of the Co(II) of cob(II)alamin and an organic radical intermediate formed during catalysis gives EPR spectra. The high-field doublet and the low-field broad signals arise from the weak interaction of an organic radical with the low-spin Co(II) of cob(II)alamin. To characterize the organic radical intermediate in the diol dehydratase reaction, several deuterated and (13)C-labeled 1,2-propanediols were synthesized, and the EPR spectra observed in the catalysis were measured using them as substrate. The EPR spectra with the substrates deuterated on C1 showed significant line width narrowing of the doublet signal. A distinct change in the hyperfine coupling was seen with [1-(13)C]-1,2-propanediol, but not with the [2-(13)C]-counterpart. Thus, the organic radical intermediate observed by EPR spectroscopy was identified as the 1,2-propanediol-1-yl radical, a C1-centered substrate-derived radical.     

An electron paramagnetic resonance study on the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydrase by glycerol and other substrates

Adenosylcobalamin-dependent diol dehydrase undergoes mechanism-based inactivation by glycerol or other substrates during catalysis. X-band electron paramagnetic resonance spectra of holoenzyme were measured at −130°C after reaction with such substrates. After short time of incubation, broad signals assigned to low-spin Co(II) of cob(II)alamin and doublet signals assigned to an organic radical intermediate derived from each substrate were observed with 1,2-propanediol, 1,2-ethanediol, glycerol and meso-2,3-butanediol with the magnitude of their exchange interaction (J-value) decreasing in this order. A substrate with the smaller magnitude of exchange interaction between low-spin Co(II) and an organic radical intermediate seems to be an efficient mechanism-based inactivator. Since the magnitude of exchange interaction decreases with the distance between radical species in a radical pair, these results suggest that a stabilizing effect of holoenzyme on radical intermediates during reactions decreases with the distance between Co(II) and a radical.     

Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase

Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13C]ethanolamine, [2-13C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13C hyperfine splitting in EPR spectra of samples prepared with [1-13C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence of a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke, K., et al. (1999) J. Am. Chem. Soc. 121, 10522-10528]. The EPR signals of the substrate radical intermediate are altered by electron spin coupling to the other paramagnetic species, cob(II)alamin, in the active site. The dipole-dipole and exchange interactions as well as the 1-13C hyperfine splitting tensor were analyzed via spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co2+-radical distance of 8.7 A was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13C hyperfine splitting tensor shows that the long axis of the spin-bearing p orbital on C1 of the substrate radical makes an angle of approximately 98 degrees with the unique axis of the d(z2) orbital of Co2+.     

The positions of radical intermediates in the active sites of adenosylcobalamin-dependent enzymes

The radical intermediates generated during the catalytic cycles of adenosylcobalamin-dependent enzymes occur in pairs. The positions of radicals residing on the cofactor, substrate or protein, relative to the position of the low-spin Co(2+) from the cob(II)alamin intermediate, can be extracted from electron paramagnetic resonance (EPR) spectra of the spin-coupled pairs. Examples of radical-Co(2+) pairs that span a range of interspin distances from 3 to 13A have been presented. Interspin distances greater than 5A require motion of one or more of the participating species. EPR spectroscopy provides a convenient means to determine the structures of these transient intermediates.     

A physical explanation of the EPR spectrum observed during catalysis by enzymes utilizing coenzyme B12.

We have proposed that the "doublet" EPR spectra observed during catalysis by a number of coenzyme B12-requiring enzymes arises from a weak electrostatic exchange interaction between an organic free radical and low spin Co(II), B12r. By varying the magnitude of the exchange of coupling we have quite accurately simulated the published EPR spectra from the enzyme systems: diol dehydrase, glycerol dehydrase, ribonucleotide reductase, and ethanolamine ammon-ia lyase. A dipolar model was shown to be incompatible with the observed properties of these systems.     

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.     

Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme.

A series of 16 analogs of 5'-deoxy-5'-adenosylcobalamin (adenosylcobalamin) were examined for their effects on the diol dehydrase system of Klebsiella pneumoniae (Aerobacter Aerogenes). Four analogs, ara-adenosyl-, aristeromycyl-, 3-isoadenosyl-, and nebularylcobalamin, were able to function as coenzymes in the diol dehydrase reaction, coenzyme activity decreasing in that order. Like the native holoenzyme, complexes of the enzyme with these four analogs show a cob(II)alamin-like absorption peak or shoulder in the presence of 1,2-propanediol. Analogs containing hypoxanthine, cytosine, or benzimidazole do not function as coenzymes, but are weak competitive inhibitors in the presence of adenosylcobalamin. Analogs in which the D-ribosyl moiety is replaced by L-ribose or by an alkyl chain of 2 to 6 carbons are inactive as coenzymes, but act as competitive inhibitors with extremely high affinity for the apoenzyme. Complexes with the inactive analogs showed visible spectra similar to those of the corresponding free cobalamins. Upon anaerobic photolysis and subsequent aeration, complexes with the first group of inactive analogs produced unusually stabilized cob(II)alamin, while complexes with the second group of inactive analogs were readily photolyzed to a hydroxocobalamin-enzyme complex. Complexes with adeninylpentyl- and L-adenosylcobalamin were stable to light under the same conditions. These findings suggest that both the ribose and the adenine moiety of the nucleoside participate in enzyme-coenzyme interaction, involving not only the binding to the apoenzyme but also the activation of the carbon-cobalt bond.     

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.     

Characterization of an allylic analogue of the 5'-deoxyadenosyl radical: an intermediate in the reaction of lysine 2,3-aminomutase.

An allylic analogue of the 5'-deoxyadenosyl radical has been characterized at the active site of lysine 2,3-aminomutase (LAM) by electron paramagnetic resonance (EPR) spectroscopy. The anhydroadenosyl radical, 5'-deoxy-3',4'-anhydroadenosine-5'-yl, is a surrogate of the less stable 5'-deoxyadenosyl radical, which has never been observed but has been postulated to be a radical intermediate in the catalytic cycles of a number of enzymes. An earlier communication [Magnusson, O.Th., Reed, G. H., and Frey, P. A. (1999) J. Am. Chem. Soc. 121, 9764-9765] included the initial spectroscopic identification at 77 K of the radical, which is formed upon replacement of S-adenosylmethionine by S-3',4'-anhydroadenosylmethionine as a coenzyme for LAM. The electron paramagnetic resonance spectrum of the radical changes dramatically between 77 and 4.5 K. This unusual temperature dependence is attributed to a spin-spin interaction between the radical and thermally populated, higher spin states of the [4Fe-4S]+2 center, which is diamagnetic at 4.5 K. The EPR spectra of the radical at 4.5 K have been analyzed using isotopic substitutions and simulations. Analysis of the nuclear hyperfine splitting shows that the unpaired spin is distributed equally between C5'- and C3'- as expected for an allylic radical. Hyperfine splitting from the beta-proton at C-2'(H) shows that the dihedral angle to the p(z)-orbital at C-3' is approximately 37 degrees. This conformation is in good agreement with a structural model of the radical. The rate of formation of the allylic radical shows that it is kinetically competent as an intermediate. Measurements of 2H kinetic isotope effects indicate that with lysine as the substrate, the rate-limiting steps follow initial reductive cleavage of the coenzyme analogue.     

Probing interactions from solvent-exchangeable protons and monovalent cations with the 1,2-propanediol-1-yl radical intermediate in the reaction of dioldehydrase

The reaction of adenosylcobalamin-dependent dioldehydrase with 1,2-propanediol gives rise to a radical intermediate observable by EPR spectroscopy. This reaction requires a monovalent cation such as potassium ion. The radical signal arises from the formation of a radical pair comprised of the Co(II) of cob(II)alamin and a substrate-related radical generated upon hydrogen abstraction by the 5'-deoxyadenosyl radical. The high-field asymmetric doublet arising from the organic radical has allowed investigation of its composition and environment through the use of EPR spectroscopic techniques. To characterize the protonation state of the oxygen substituents in the radical intermediate, X-band EPR spectroscopy was performed in the presence of D(2)O and compared to the spectrum in H(2)O. Results indicate that the unpaired electron of the steady-state radical couples to a proton on the C(1) hydroxyl group. Other spectroscopic experiments were performed, using either potassium or thallous ion as the activating monovalent cation, in an attempt to exploit the magnetic nature of the (205,203)Tl nucleus to identify any intimate interaction of the radical intermediate with the activating cation. The radical intermediate in complex with dioldehydrase, cob(II)alamin and one of the activating monovalent cations was observed using EPR, ENDOR, and ESEEM spectroscopy. The spectroscopic evidence did not implicate a direct coordination of the activating cation and the substrate derived radical intermediate.     

Mechanism of action of adenosylcobalamin: glycerol and other substrate analogues as substrates and inactivators for propanediol dehydratase--kinetics, stereospecificity, and mechanism.

A number of vicinal diols were found to react with propanediol dehydratase, typically resulting in the conversion of enzyme-bound adenosylcobalamin to cob(II)alamin and formation of aldehyde or ketone derives from substrate. Moreover, all are capable of effecting the irreversible inactivation of the enzyme. The kinetics and mechanism of product formation and inactivation were investigated. Glycerol, found to be a very good substrate for diol dehydratase as well as a potent inactivator, atypically, did not induce cob(II)alamin formation to any detectable extent. With glycerol, the inactivation process was accompanied by conversion of enzyme-bound adenosylcobalamin to an alkyl or thiol cobalamin, probably by substitution of an amino acid chain near the active site for the 5'-deoxy-5'-adenosyl ligand on the cobalamin. The inactivation reaction with glycerol as the inactivator exhibits a deuterium isotope effect of 14, strongly implicating hydrogen transfer as an important step in the mechanism of inactivation. The isotope effect on the rate of product formation was found to be 8.0. Experiments with isotopically substituted glycerols indicate that diol dehydrase distinguishes between "R" and "S" binding conformations, the enzyme-(R)-glycerol complex being predominately responsible for the product-forming reaction, while the enzyme-(S)-glycerol complex results primarily in the activation reaction. Mechanistic implications are discussed. A method for removing enzyme-bound hydroxycobalamin that is nondestructive to the enzyme and a technique for measuring the binding constants of (R)- and (S)-1,2-propanediols are presented.     

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.     

The binding site for the adenosyl group of coenzyme B12 in diol dehydrase

The binding of cob(II)alamin (CblII) and 5'-deoxyadenosine to diol dehydrase was studied spectroscopically and with [U-14C]5'-deoxyadenosine. CblII was bound to this enzyme forming a tight 1:1 complex which was resistant to oxidation by O2 even in the presence of CN-. An irreversible 1:1:1 ternary complex was formed between enzyme, CblII, and 5'-deoxyadenosine, when the enzyme was incubated first with the nucleoside and then with CblII. When this order of addition of the constituents was reversed, no 5'-deoxyadenosine was bound to the enzyme-CblII complex. Hydroxocobalamin could also bind to the enzyme together with the nucleoside, while other cob(III)alamins bearing a bulkier Co beta ligand displaced the nucleoside upon binding to the enzyme. The binding of [U-14C]5'-deoxyadenosine was strongly inhibited by unlabeled 5'-deoxy-ara-adenosine, 4',5'-anhydroadenosine, adenosine, adenine, and 5',8-cyclic adenosine, in this order, but not by 5'-deoxyuridine. These results constitute direct evidence for the presence of the binding site for the adenosyl group of adenosylcobalamin, which is spatially limited to and highly specific for adenine nucleosides. The binding of 5'-deoxyadenosine to the apoenzyme was reversible.     

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 adenosylcobalamin-dependent glutamate mutase with 2-thiolglutarate

We have investigated the reaction of glutamate mutase with the glutamate analogue, 2-thiolglutarate. In the standard assay, 2-thiolglutarate behaves as a competitive inhibitor with a Ki of 0.05 mM. However, rather than simply binding inertly at the active site, 2-thiolglutarate elicits cobalt-carbon bond homolysis and the formation of 5'-deoxyadenosine. The enzyme exhibits a complicated EPR spectrum in the presence of 2-thiolglutarate that is markedly different from any previously observed with the enzyme. The spectrum was simulated well by assuming that it arises from electron-electron spin coupling between a thioglycolyl radical and low-spin Co2+ in cob(II)alamin. Analysis of the zero-field splitting parameters obtained from the simulations places the organic radical approximately 10 A from the cobalt and at a tilt angle of approximately 70 degrees to the normal of the corrin ring. This orientation is in good agreement with that expected from the crystal structure of glutamate mutase complexed with the substrate. 2-Thiolglutarate appears to react in a manner analogous to that of glutamate by first forming a thiolglutaryl radical at C-4 that then undergoes fragmentation to produce acrylate and the sulfur-stabilized thioglycolyl radical. The thioglycolyl radical accumulates on the enzyme, suggesting it is too stable to undergo further steps in the mechanism at a detectable rate.     

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.     

Enhancing the methyl-donor activity of methylcobalamin by covalent attachment of DNA

The preparation of a covalent DNA conjugate of vitamin B12 by means of heterogeneous solid-phase synthesis is reported. The cyano-corrinoid made available, dipotassium Co(beta)-cyanocobalamin-(3'-->2'),(3'-->5')-bis-2'-deoxythymidyl-3'-ate (K(2)-4), was cleanly methylated at the Co center by electrosynthetic means. Aqueous solutions of the resulting organometallic DNA-B12 conjugate K(2)-5 exhibited spectroscopic properties indicative of significant weakening of the axial (Co-N) bond, together with a 25-times higher basicity relative to Co(beta)-methylcobalamin (2). Methyl-transfer equilibria of pH-neutral aqueous solutions of K(2)-5 and cob(I)alamin (K-7) on one side, and of cob(I)alamin-(3'-->2'),(3'-->5')-bis-2'-deoxythymidyl-3'-ate (K(3)-8) and methylcobalamin (2) on the other, were studied at room temperature (Scheme 3). The NMR-derived data provided an equilibrium constant of ca. 0.3. Activation of K(2)-5 for abstraction of its Co-bound Me group by a nucleophile (such as cob(I)alamin) was, thus, indicated.     

The synthesis of adenine-modified analogs of adenosylcobalamin and their coenzymic function in the reaction catalyzed by diol dehydrase

Five analogs of adenosylcobalamin modified in the adenine moiety of the Co beta ligand were synthesized and tested for coenzymic function with diol dehydrase of Klebsiella pneumoniae ATCC 8724. 1-Deaza and 3-deaza analogs of adenosylcobalamin were active as coenzyme, whereas 7-deaza and N6,N6-dimethyl derivatives and guanosylcobalamin did not show detectable coenzymic activity. 7-Deaza and N6,N6-dimethyl analogs acted as strong competitive inhibitors with respect to adenosylcobalamin. The formation of cob(II)alamin as intermediate in the catalytic reaction was spectroscopically observed with catalytically active complexes of the enzyme with 1-deaza and 3-deaza analogs in the presence of 1,2-propanediol, but not with complexes with the inactive analogs. Oxygen sensitivity of the enzyme-analog complexes suggests that the carbon-cobalt bond of 1-deaza and 3-deaza analogs becomes activated by the enzyme even in the absence of substrate. These results indicate that the importance of the nitrogen atoms in the adenine moiety of the coenzyme for manifestation of catalytic function and for activation of the carbon-cobalt bond decreases in the following order: N-7 greater than 6-NH2 greater than N-3 greater than N-1. The dissociation constant for 5'-deoxyadenosine determined by equilibrium dialysis at 37 degrees C was about 23 microM.