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.     

Roles of the D-ribose and 5,6-dimethylbenzimidazole moieties of the nucleotide loop of adenosylcobalamin in manifestation of coenzymic function in the diol dehydrase reaction

Coenzyme analogs in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analog containing imidazole instead of 5,6-dimethylbenzimidazole were synthesized. Coordination of the 5,6-dimethylbenzimidazole to the cobalt atom in these analogs was much weaker than that in cobalamins. The replacement of this base with imidazole did not significantly alter the strength of the coordination to the cobalt atom. 5,6-Dimethylbenzimidazolyl trimethylene and tetramethylene and imidazolyl trimethylene analogs were partially active as coenzymes in the diol dehydrase reaction in this order as judged by kcat, but the others were not active as coenzymes and were weak competitive inhibitors. This indicates that neither the alpha-D-ribofuranose ring nor the functional groups of the ribose moiety are essential for coenzymic function. There was an optimum loop size of the analogs for catalysis and for tight binding to the apoenzyme, which corresponds to the loop size of cobalamins. Therefore, the D-ribose moiety seems important as a spacer to keep the base in the proper position. The reaction with the imidazolyl trimethylene analog as coenzyme was accompanied with concomitant rapid inactivation during catalysis. The inactivation occurred only in the presence of substrate. Upon inactivation with this analog, 5'-deoxyadenosine and a B12r-like species were formed from the adenosyl group and the rest of the analog molecule, respectively, without modification of the apoenzyme. Therefore, it can be concluded that this is a kind of suicide inactivation which occurred from one of the intermediates in the normal catalytic process. The dimethylbenzo moiety of the regular coenzyme thus seems to play an important role in preventing the intermediate complexes from inactivation during catalysis.     

Roles of β-d-ribofuranose ring and the functional groups of the d-ribose moiety of adenosylcobalamin in the diol dehydratase reaction

Four analogs of adenosylcobalamin (AdoCbl) modified in the d-ribose moiety of the Coβ ligand were synthesized, and their coenzyme properties were studied with diol dehydratase of Klebsiella pneumoniae ATCC 8724. 2′-Deoxyadenosylcobalamin (2′-dAdoCbl) and 3′-deoxyadenosylcobalamin (3′-dAdoCbl) were active as coenzyme. 2′,3′-Secoadenosylcobalamin (2′,3′-secoAdoCbl), an analog bearing the same functional groups as AdoCbl but nicked between the 2′ and 3′ in the ribose moiety, and its 2′,3′-dialdehyde derivative (2′,3′-secoAdoCbl dialdehyde) were totally inactive analogs of the coenzyme. It is therefore evident that the β-d-ribofuranose ring itself, possibly its rigid structure, is essential and much more important than the functional groups of the ribose moiety for coenzyme function (relative importance; β-d-ribofuranose ring ⪢ 3′-OH ⪢ 2′-OH ⪢ ether group). With 2′-dAloCbl and 3′-dAdoCbl as enzymes. an absorption peak at 478 nm appeared during enzymatic reaction, suggesting homolysis of the CCo bound to form cob(II)alamin as intermediate. In the absence of substrate, the complexes of the enzyme with these active analogs underwent rapid inactivation by oxygen. This suggests that their CCo bond is activated even in the absence of substrate by binding to the apoprotein. No significant spectral changes were observed with 2′,3′-secoAdoCbl upon binding to the apoenzyme. In contrast, spectroscopic observation indicates that 2′,3′-secoAdoCbl dialdehyde, another inactive analog, underwent gradual and irreversible cleavage of the CCo bond by interaction with the apodiol dehydratase, forming the enzyme-bound cob(II)alamin without intermediates.     

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.     

Studies on the mechanism of adenosylcobalamin-dependent ribonucleotide reduction by the use of analogs of the coenzyme.

A series of 17 analogs of 5'-deoxy-5'-adenosylcobalamin(adenosylcobalamin) have been synthesized with modifications in the base or ribose moiety of the nucleoside ligand. These analogs have been examined for their effects on reactions catalyzed by the ribonucleotide reductase of Lactobacillus leichmannii. All the analogs are inhibitors of ATP reduction in the presence of adenosylcobalamin as coenzyme, and hence all are bound to the catalytic site. Only the 3-beta-D-ribofuranosyladenine analog (isoadenosylcobalamin) showed substantial activity as a coenzyme in ATP reduction, giving a rate of 59% of that obtained with the adenosylcobalamin. Lesser rates of reduction were obtained with nebularyl-, 2'-deoxyadenosyl-, tubercidyl-, isopropylideneadenosyl-, L-adenosyl-, and ara-adenosylcobalamin, coenzyme activity decreasing in that order. Other analogs showed no significant coenzyme activity. The rate of hydrogen exchange into water from the 5'-methylene group of the nucleoside ligand appeared to parallel the coenzyme activity in those analogs examined, but only the four cobalamins with highest coenzyme activity (adenosyl, isoadenosyl, nebularyl, 2'-deoxyadenosyl) gave detectable amounts of "active coenzyme B12," THe rapidly formed paramagnetic intermediate of ribonucleotide reduction. The enzyme system produced the slowly formed paramagnetic species characterized by a doublet EPR spectrum only with adenosyl- and isoadenosylcobalamin. By contrast the enzymic degradation of analogs to cob(II)alamin and 5'-deoxynucleoside occurred not only with those analogs active as coenzymes and in the exchange reaction but also with a number of coenzymically inactive analogs, and the rate of degradation was unrelated to the rate of ribonucleotide reduction for those analogs with coenzyme activity.     

Functions of the D-ribosyl moiety and the lower axial ligand of the nucleotide loop of coenzyme B(12) in diol dehydratase and ethanolamine ammonia-lyase reactions

The roles of the D-ribosyl moiety and the bulky axial ligand of the nucleotide loop of adenosylcobalamin in coenzymic function have been investigated using two series of coenzyme analogs bearing various artificial bases. The 2-methylbenzimidazolyl trimethylene analog that exists exclusively in the base-off form was a totally inactive coenzyme for diol dehydratase and served as a competitive inhibitor. The benzimidazolyl trimethylene analog and the benzimidazolylcobamide coenzyme were highly active for diol dehydratase and ethanolamine ammonia-lyase. The imidazolylcobamide coenzyme was 59 and 9% as active as the normal coenzyme for diol dehydratase and ethanolamine ammonia-lyase, respectively. The latter analog served as an effective suicide coenzyme for both enzymes, although the partition ratio (k(cat)/k(inact)) of 630 for ethanolamine ammonia-lyase is much lower than that for diol dehydratase. Suicide inactivation was accompanied by the accumulation of a cob(II)amide species, indicating irreversible cleavage of the coenzyme Co-C bond during the inactivation. It was thus concluded that the bulkiness of a Co-coordinating base of the nucleotide loop is essential for both the initial activity and continuous catalytic turnovers. Since the k(cat)/k(inact) value for the imidazolylcobamide in diol dehydratase was 27-times higher than that for the imidazolyl trimethylene analog, it is clear that the ribosyl moiety protects the reaction intermediates from suicide inactivation. Stopped-flow measurements indicated that the rate of Co-C bond homolysis is essentially unaffected by the bulkiness of the Co-coordinating base for diol dehydratase. Thus, it seems unlikely that the Co-C bond is labilized through a ground state mechanochemical triggering mechanism in diol dehydratase.     

Acceleration of cleavage of the carbon-cobalt bond of sterically hindered alkylcobalamins by binding to apoprotein of diol dehydrase

Cleavage of the C-Co bond of sterically hindered alkylcobalamins bearing neither an adenine moiety nor functional groups, such as isobutylcobalamin, neopentylcobalamin, and cyclohexylcobalamin, was markedly accelerated by their interaction with apoprotein of diol dehydrase, although these cobalamins do not function as coenzyme. Acceleration of the conversion of alkylcobalamins to enzyme-bound hydroxocobalamin was stoichiometric and obeyed first-order reaction kinetics. These results, together with strong competitive inhibition by these alkylcobalamins with respect to adenosylcobalamin, indicate that acceleration of the C-Co bond cleavage by the apoenzyme is due to labilization of their C-Co bond by binding to the active site of the enzyme. This labilization is considered to be caused by a steric distortion of the corrin ring which is induced by specific tight interaction of the cobalamin moiety with apoprotein. The importance of such a labilizing effect for activation of the C-Co bond of adenosylcobalamin in enzymatic reactions is discussed.     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

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.     

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.     

Activation and cleavage of the carbon-cobalt bond of adeninylethylcobalamin by diol dehydrase

Adeninylethylcobalamin (AdeEtCbl) underwent cleavage of the C-Co bond by interaction with apoprotein of diol dehydrase from Klebsiella pneumoniae ATCC 8724, although this analog was quite inactive as coenzyme. Spectroscopic observation indicates that AdeEtCbl was converted to the enzyme-bound hydroxocobalamin without intermediates. The conversion was stoichiometric (1:1) and obeyed the second-order reaction kinetics (k = 0.027 min-1 microM-1 at 37 degrees C) depending upon concentrations of apoprotein and AdeEtCbl. This suggests that the complex formation is the rate-determining step and that AdeEtCbl undergoes rapid C-Co bond cleavage once it binds to the apoenzyme. Substrates and oxygen did apparently not affect the rate of the C-Co bond cleavage. The experiments using [adenine-U-14C]AdeEtCbl and [1(3)-3H]glycerol demonstrated that 9-ethyladenine was the only product formed from the adeninylethyl group of AdeEtCbl during the conversion and that an additional hydrogen atom in the 9-ethyladenine is not derived from the substrate. 1H NMR measurement of the 9-ethyladenine formed enzymatically from AdeEtCbl and DL-1,2-[1,1,2-2H3]propanediol also led to the same conclusion. All of these results indicate that the C-Co bond of AdeEtCbl is activated by diol dehydrase and undergoes heterolysis forming Co(III) and a carbanion or a carbanion-like species, in clear contrast to the homolysis of the C-Co bond of adenosylcobalamin in the normal catalytic process. 9-Ethyladenine formed remained tightly associated with the enzyme. Longer chain homologs, i.e. adeninylpropylcobalamin, adeninylbutylcobalamin, and adeninylpentylcobalamin did not undergo such cleavage of the C-Co bond by diol dehydrase.     

Chemical modification of coenzyme B12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal-P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH₄. ?-N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH₄-reduced enzyme-pyridoxal-P complex. The relationship of inactivation vs pyridoxal-P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal-P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal-P results from irreversible dissociation of the enzyme into subunits upon pyridoxal-P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal-P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).     

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.     

Mechanism of reactivation of coenzyme B12-dependent diol dehydratase by a molecular chaperone-like reactivating factor.

The mechanism of reactivation of diol dehydratase by its reactivating factor was investigated in vitro by using enzyme. cyanocobalamin complex as a model for inactivated holoenzyme. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins through intermediate formation of apoenzyme. The factor showed extremely low but distinct ATP-hydrolyzing activity. It formed a tight complex with apoenzyme in the presence of ADP but not at all in the presence of ATP. Incubation of the enzyme.cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin, leaving the tight apoenzyme-reactivating factor complex. Although the resulting complex was inactive even in the presence of added adenosylcobalamin, it dissociated by incubation with ATP, forming the apoenzyme, which was reconstitutable into active holoenzyme with added coenzyme. Thus, it was established that the reactivation of the inactivated holoenzyme by the factor in the presence of ATP and Mg2+ takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme.factor complex. ATP plays dual roles as a precursor of ADP in the first step and as an effector to change the factor into the low-affinity form for diol dehydratase. The enzyme-bound adenosylcobalamin was also susceptible to exchange with free adeninylpentylcobalamin, although to a much lesser degree. The mechanism for discrimination of adenine-containing cobalamins from adenine-lacking cobalamins was explained in terms of formation equilibrium constants of the cobalamin.enzyme.reactivating factor ternary complexes. We propose that the reactivating factor is a new type of molecular chaperone that participates in reactivation of the inactivated enzymes.     

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.     

Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol

The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co-C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5'-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg(2+) and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5'-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H(2)S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.     

Thiol groups of chicken liver LDH (author's transl)]

Crystallized chicken liver H4 lactatedehydrogenase with PCBM and DTNB, proved to have sic thiol groups per enzyme molecule. Sulphydryl groups seemed necessary for activity since the enzyme became inactive when the groups were blocked by PCMB, DTNB or by Zn (II), Cu (II) or Hg (II). LDH inhibited by Hg (II) recovered its activity after treatment with beta-mercaptoentanol. LDH reversible inactivation, caused by PCMB, was partially impeded by NAD, NADH hand L-lactate but inactivation caused by DTNB was impeded in any way by coenzymes or substrates. PCMB is a competitive inhibitor with the coenzymes but is non-competitive with the substrates whereas DTNB is a competitive inhibitor with NADH or L-lactate. Kinetic studies of the DTNB inactivation suggest the possible formation of a DTNB-LDH-NADH complex. The formation of LDH-NADH and LDH-NAD pyruvate inactive complexes have been detected by U.V. absorbancy measurements. Such inactive complexes have equally been observed experimenting with the PCMB of Hg (II) previously treated enzyme. The results showed that these essential sulphydryl groups are not involved in th attaching of coenzymes or substrates to the chicken liver LDH molecule, but they seem to suggest the participation of --SH groups during the reversible hydrogen transfer between NADH and pyruvate.     

Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase

Rat methionine synthase was expressed chiefly as apoenzyme in recombinant baculovirus-infected insect cells (Yamada, K., Tobimatsu, T., and Toraya, T. (1998) Biosci. Biotech. Biochem. 62, 2155-2160). The apoenzyme produced was very unstable, and therefore, after complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K(m) values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob(II)alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium(III) and S-adenosylmethionine. The rate of oxidative inactivation of the enzyme in the absence of S-adenosylmethionine was slower with a stronger reducing agent like titanium(III). The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective or were effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme, which was convertible to holoenzyme by photoirradiation.     

The synthesis and properties of four spin-labeled analogs of adenosylcobalamin

Four spin-labeled analogs of adenosylcobalamin have been synthesized to aid in the detection and identification of radical intermediates in the adenosylcobalamin-dependent enzymatic reactions and to serve as probes of the coenzyme, substrate, and effector binding sites of the protein. Three isomers of adenosylcobalamin, in which one of the propionamide side chains (b, d, or e) was hydrolyzed, and adenosylepicobalamin e-carboxylic acid were reacted with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to yield the spin-labeled adenosylcorrinoids. These spin-labeled derivatives of adenosylcobalamin function as coenzymes and/or inhibitors of dioldehydrase from Klebsiella pneumoniae and of ribonucleotide reductase from Corynebacterium nephridii. Electron spin resonance has been used to monitor the photolytic cleavage of the carbon-cobalt bond of these analogs.     

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.