Báo cáo khoa học: Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase docx

Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B 12 -dependent diol dehydratase and ethanolamine ammonia-lyase Masaki Fukuoka 1 , Yuka Nakanishi 1 , Renate B. Hannak 2 , Bernhard Kra ¨ utler 2 and Tetsuo Toraya 1 1 Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama, Japan 2 Institut fu ¨ r Organische Chemie, Universita ¨ t Innsbruck, Innsbruck, Austria AdoCbl participates as coenzyme for the enzymes that catalyze carbon skeleton rearrangements, heteroatom eliminations, and intramolecular amino group migra- tions [1–3]. For example, diol dehydratase (EC 4.2.1.28) and ethanolamine ammonia-lyase (EC 4.3.1.7) catalyze the dehydration of 1,2-diols and the deamination of eth- anolamine to the corresponding aldehydes, respectively [4–6]. These reactions proceed by a radical mechanism, and an essential early event in all the AdoCbl-dependent rearrangements is the generation of a catalytic radical (adenosyl radical) by homolytic cleavage of the coen- zyme’s Co-C bond [1,7]. Recently, the X-ray structures of several AdoCbl- dependent enzymes have been solved in complexes with cobalamins [8–13]. Spatial isolation of the radical intermediates in the active site cavity seems to be the common strategy for the so-called ‘negative catalysis’ of the Re ´ tey’s concept [14]. The interactions of the coenzyme’s adenine moiety with enzymes were revealed with methylmalonyl-CoA mutase [15,16], diol Keywords adenosylcobalamin; coenzyme B 12 ; homoadenosylcobalamins; diol dehydratase; ethanolamine ammonia-lyase Correspondence T. Toraya, Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700–8530, Japan Fax: +81 86 2518264 Tel: +81 86 2518194 E-mail: toraya@cc.okayama-u.ac.jp (Received 11 May 2005, revised 20 July 2005, accepted 1 August 2005) doi:10.1111/j.1742-4658.2005.04892.x [x-(Adenosyl)alkyl]cobalamins (homoadenosylcobalamins) are useful ana- logues of adenosylcobalamin to get information about the distance between Co and C5¢, which is critical for Co-C bond activation. In order to use them as probes for exploring the active sites of enzymes, the coenzymic properties of homoadenosylcobalamins for diol dehydratase and ethanol- amine ammonia-lyase were investigated. The k cat and k cat ⁄ K m values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regu- lar coenzyme with diol dehydratase, respectively. The k cat ⁄ k inact value showed that the holoenzyme with this analogue becomes inactivated on average after about 3000 catalytic turnovers, indicating that the probability of inactivation during catalysis is almost 500 times higher than that for the regular holoenzyme. The k cat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonia- lyase, as judged from the initial velocity, but the holoenzyme with this ana- logue underwent inactivation after on average about 50 catalytic turnovers. This probability of inactivation is 3800 times higher than that for the regu- lar holoenzyme. When estimated from the spectra of reacting holoenzymes, the steady state concentration of cob(II)alamin intermediate from aden- osylmethylcobalamin was very low with either diol dehydratase or ethanol- amine ammonia-lyase, which is consistent with its extremely low coenzymic activity. In contrast, neither adenosylethylcobalamin nor adeninylpentylco- balamin served as active coenzyme for either enzyme and did not undergo Co-C bond cleavage upon binding to apoenzymes. Abbreviations AdoCbl, adenosylcobalamin or coenzyme B 12 ; AdoEtCbl, adenosylethylcobalamin; AdoMeCbl, adenosylmethylcobalamin or homocoenzyme B 12 ; AdePeCbl, adeninylpentylcobalamin. FEBS Journal 272 (2005) 4787–4796 ª 2005 FEBS 4787 dehydratase [17], glutamate mutase [18], and lysine 5,6-aminomutase [13]. Based on the structures, steric strain models of activation and cleavage of the Co-C bond were proposed [17,18]. In diol dehydratase, tight interactions between the enzyme and coenzyme at both the cobalamin moiety and the adenine ring of the adenosyl group seem to produce angular strains and tensile force that likely contribute to labilization of the Co-C bond [17] (Fig. 1A). Difference in the specificity for the adenosyl group among enzymes may reflect the difference of the adenosyl group-bind- ing sites of the enzymes. From simulation of the EPR spectra of reacting holoenzymes, it was suggested that that the distances between Co(II) of cob(II)alamin and substrate radicals are  11 A ˚ in ethanolamine ammonia-lyase [19,20], ‡ 10 A ˚ in diol dehydratase [21], 6.6 A ˚ in glutamate mutase [22], and 6.0 A ˚ in methylmalonyl-CoA mutase [23]. These suggestions were corroborated by the X-ray structures of glutam- ate mutase [10], methylmalonyl-CoA mutase [8], diol dehydratase [9], and glycerol dehydratase [12]. The difference in the distances between substrate radical and Co(II) may cause different ways of radical trans- fer from coenzyme to substrate. For the access to substrates, ribosyl rotation (Fig. 1A) and pseudorota- tion models of this process have been proposed for A B Fig. 1. Modeling studies of diol dehydratase on adenosyl radical formation and access to substrate (A) and partial structures of AdoCbl ana- logues used in this study (B). (A) The steric strain model of the Co-C bond cleavage by diol dehydratase and the ribosyl rotation model of access of the adenosyl group to substrate [17]. Left, Superimposition of AdoCbl over that of enzyme-bound AdePeCbl at the cobalamin moi- ety without cleavage of the Co-C bond. Center, The same superimposition at both the cobalamin moiety and the adenine ring with the Co-C bond cleaved and the Co-C distance kept at a minimum (‘proximal’ conformation). Right, Superimposed with the ribose moiety of the adeno- syl group rotated around the glycosidic linkage so that C5¢ is closest to C1 of the substrate (‘distal’ conformation). Stick model represents the adenosyl group of AdoCbl. Residue numbers in the a subunit. (B) Partial structures of AdoCbl analogues used in this study. R represents the Cob (upper axial) ligand. Coenzymic functions of homoadenosylcobalamins M. Fukuoka et al. 4788 FEBS Journal 272 (2005) 4787–4796 ª 2005 FEBS diol dehydratase and glutamate mutase, respectively [17,18]. It would be beneficial to get information about the distance between Co and C5¢, which is critical for the Co-C bond activation, especially for the enzymes whose X-ray structures are not yet available. A series of [x-(adenosyl)alkyl]cobalamins, i.e. homoadenosyl- cobalamins (Fig. 1B), have been synthesized [24–26]. These analogues might be useful as probes for explor- ing the active sites of AdoCbl-dependent enzymes. In this paper, we have investigated the coenzymic proper- ties of these homologues for diol dehydratase and ethanolamine ammonia-lyase. Results Coenzymic activity of the coenzyme analogues in the diol dehydratase and ethanolamine ammonia-lyase reactions Coenzymic activity of homoadenosylcobalamins was first examined using AdoCbl-dependent diol dehydra- tase as a test enzyme. Figure 2A indicates the time cour- ses of the diol dehydratase reaction using AdoMeCbl and AdoEtCbl at a concentration of 10 lm. When 300 times higher concentration of apoenzyme than that for AdoCbl was used, low but distinct coenzyme activity was observed with AdoMeCbl. As shown in Table 1, its k cat value was about 0.27% that for AdoCbl, and the rate of mechanism-based inactivation (k inact ) with this analogue was as slow as that with the regular coenzyme. As judged from the k cat ⁄ k inact value, the holoenzyme with this analogue becomes inactivated after about 3000 catalytic turnovers on average. This indicates that the probability of inactivation during catalysis is almost 500 times higher than that with AdoCbl. The catalytic efficiency (k cat ⁄ K m ) of the holoenzyme with AdoMeCbl was 0.15% that for the regular coenzyme. Fig. 2. Time courses of reactions with homoadenosylcobalamins as coenzymes. Propionaldehyde and acetaldehyde formed by enzy- matic reactions were assayed by the alcohol dehydrogenase-NADH coupled method, as described in the text. The amounts of apo- enzyme used are given below in parentheses. Reactions were initi- ated by adding each coenzyme at a concentration of 10 l M. (A) Diol dehydratase reaction. AdoCbl (solid line) (0.01 unit); AdoMeCbl (dashed line) (3 units). (B) Ethanolamine ammonia-lyase reac- tion. AdoCbl (solid line) (0.01 unit); AdoMeCbl (dashed line) (34 units). Table 1. Coenzyme activity and kinetic parameters for the analogues in the diol dehydratase and ethanolamine ammonia-lyase reactions (determined at 37 °C). Coenzyme Diol dehydratase Ethanolamine ammonia-lyase k cat b k inact b (min )1 ) k cat ⁄ k inact c · 10 )4 K m c (lM) k cat ⁄ K m · 10 )6 (M )1 Æs )1 ) K i c (lM) k cat b k inact b (min )1 ) k cat ⁄ k inact · 10 )4 (s )1 )(%) (s )1 )(%) AdoCbl a 366 (100) 0.014 157 0.80 458 447 (100) 0.14 19 AdoMeCbl 1.0 (0.3) 0.022 0.3 1.7 ± 0.3 0.6 ± 0.1 1.1 ± 0.3 0.6 e (0.1) 0.64 0.006 AdoEtCbl  0.0 ( 0.0) 1.5 ± 0.5  0.0 ( 0.0) AdePeCbl  0.0 ( 0.0) 0.27 d  0.0 ( 0.0) a From [55]. b Determined by the alcohol dehydrogenase-NADH coupled assay method. c Determined by the MBTH method and Lineweaver– Burk plots. Averages of two independent experiments are shown. d From [27]. e From the initial velocity. M. Fukuoka et al. Coenzymic functions of homoadenosylcobalamins FEBS Journal 272 (2005) 4787–4796 ª 2005 FEBS 4789 In contrast, neither AdoEtCbl nor AdePeCbl was an active coenzyme even when examined with 3000 times higher concentration of apoenzyme. These inactive analogues behaved as strong competitive inhibitors, as judged from their inhibition constants (K i ). This fact indicates that they can not serve as coenzymes although they are bound tightly to the apoenzyme. The observation that the K i value for AdePeCbl is smaller than those for the other analogues [27] is consistent with the previous report on the effects of [x-(adenosin-5¢-O-yl)alkyl]cobalamins [28]. Coenzymic activity of the homoadenosylcobalamins was measured with AdoCbl-dependent ethanolamine ammonia-lyase as well. The time courses of the etha- nolamine ammonia-lyase reaction using AdoMeCbl and AdoEtCbl at a concentration of 10 lm are shown in Fig. 2B. Again, very low but distinct coenzymic activity was observed with AdoMeCbl when deter- mined with 1500 times higher concentration of apoenzyme, but the holoenzyme with AdoMeCbl underwent rapid inactivation. Kinetic constants shown in Table 1 indicate that the coenzymic activity (k cat )of this analogue is about 0.13% that of AdoCbl, as judged from the initial velocity. The rate of mechan- ism-based inactivation (k inact ) with this analogue was five times faster than that with the regular coenzyme. The k cat ⁄ k inact value for AdoMeCbl indicates that the holoenzyme with this analogue undergoes inactivation after about 50 catalytic turnovers on average, and that this probability of inactivation is 3800 times higher than that with AdoCbl. On the other hand, AdoEtCbl and AdePeCbl were totally inactive as coenzymes even when measured with 1500 times higher concentration of apoenzyme, in accordance with the results using diol dehydratase. Spectroscopic studies Figure 3A–D shows the spectra of free AdoCbl, its homologues and AdePeCbl, respectively. When these Fig. 3. Spectral changes of homoadenosylcobalamins upon incubation with apoenzymes of diol dehydratase and ethanolamine ammonia- lyase in the presence of substrates. Free AdoCbl (3.5 l M) (A), AdoMeCbl (3.8 lM) (B) AdoEtCbl (3.8 lM) (C), or AdePeCbl (3.8 lM)(D)in 35 m M potassium phosphate buffer (pH 8.0) containing 1 M propane-1,2-diol (solid lines). Spectra of the photolyzed analogues were also taken (broken lines). Apodiol dehydratase (100 unitsÆmL )1 ,6.4lM) was incubated with AdoCbl (3.5 lM) (E) AdoMeCbl (3.8 lM) (F), AdoEtCbl (3.8 l M) (G), or AdePeCbl (3.8 lM) (H) in 35 mM potassium phosphate buffer (pH 8.0) containing 1 M propane-1,2-diol, in a volume of 1.0 mL. Spectra were taken at 5 min of incubation (solid lines). After 10 min, the enzyme was denatured by adding 6 M guanidine. HCl ⁄ 0.06 M citric acid. The pH of the mixture was 2.6. After 10 min at 37 °C, the mixture was neutralized by adding 200 lLof1 M potassium phosphate buf- fer (pH 8.0) and 70 lLof5 M KOH, and the spectrum was taken (dotted lines). Samples were finally illuminated at 0 °C for 10 min with a 250-W tungsten light bulb at a distance of 15 cm (broken lines). Apoethanolamine ammonia-lyase (50 unitsÆmL )1 , 8.0 lM) was incubated with AdoCbl (3.5 l M) (I), AdoMeCbl (3.8 lM) (J), AdoEtCbl (3.8 lM) (K), or AdePeCbl (3.8 lM) (L) in 31 mM potassium phosphate buffer (pH 8.0) containing 0.2 M ethanolamine and 5 mM 2-mercaptoethanol, in a volume of 1.0 mL. Spectra were measured at 5 min of incubation (solid lines). Spectra of the denaturated and neutralized (dotted lines) and illuminated (broken lines) samples were measured as described above for E–H. Spectra are corrected for dilution. Coenzymic functions of homoadenosylcobalamins M. Fukuoka et al. 4790 FEBS Journal 272 (2005) 4787–4796 ª 2005 FEBS analogues of AdoCbl were incubated with apodiol dehydratase in the presence of substrate (propane-1,2- diol), the spectra of analogues underwent bathochro- mic shifts of the a-band by 10–20 nm (Fig. 3F–H) as compared with those of free counterparts. The extents of the Co-C bond cleavage of these analogues were negligible although that of AdoCbl was estimated to be  85% from Fig. 3E. Upon denaturation of the enzyme-cobalamin complexes by guanidine under aci- dic conditions, the spectra resembling free analogues were obtained. They were then changed to the spec- trum of aquacobalamin upon photoillumination. From these results, it was concluded that the steady state concentration of cob(II)alamin intermediate from AdoMeCbl is very low in consistence with its extre- mely low coenzymic activity, and that neither analogue undergoes irreversible cleavage of the Co-C bond upon binding to apoenzyme. We have reported previously that the complexes of diol dehydratase with adeninylbutylcobalamin or AdePeCbl showed resistance to photolysis of the Co-C bond as compared with the free counterparts [27]. Figure 4C,D indicates that both of the enzyme-bound AdoMeCbl and AdoEtCbl are much more resistant to photolysis of their Co-C bond upon photoillumination than the free counterparts (Fig. 4A,B). The conver- sions of the enzyme-bound analogues to OH-Cbl were approximately 10% for AdoMeCbl and 2% for AdoEtCbl, respectively, although that of the free coun- ter parts was 100% under the conditions. This suggests that the upper axial ligand-derived radicals, such as adenosylmethyl and adenosylethyl radicals, readily recombine with the cob(II)alamin intermediate to re-form original organocobalamins. Such a property of these coenzyme analogues would be reasonably explained by the fact that adenine-anchored radicals are formed by photolysis of their Co-C bond and kept associated with the active site. The adenine moiety would be trapped or anchored in the so-called ‘aden- ine-binding pocket’ of the enzyme whose structure has been analyzed by X-ray crystallography [17]. Similar experiments were carried out with ethanol- amine ammonia-lyase. Again, none of AdoMeCbl, AdoEtCbl and AdePeCbl underwent significant spec- tral changes upon binding to the enzyme in the pres- ence of ethanolamine, although they showed red or blue shift of the a-band by less than 6 nm (Fig. 3J–L). The steady-state concentrations of cob(II)alamin inter- mediate were almost negligible with the analogues, although that with the regular coenzyme was estimated to be c. 88% from Fig. 3I. This is consistent with its extremely low coenzymic activity of AdoMeCbl and with inactivity of the other two analogues. It is also evident that neither AdoEtCbl nor AdePeCbl under- goes irreversible cleavage of the Co-C bond upon bind- ing to ethanolamine ammonia-lyase. Discussion The data presented in this paper can be reasonably explained by our ‘steric strain model’ of the coen- zyme’s Co-C bond activation upon binding to apo- enzyme (Fig. 1A) [17]. AdoMeCbl [24] and AdoEtCbl [29] were reported to be totally inactive as coenzyme for ribonucleotide reductase, and the latter inactive for diol dehydratase [27]. The X-ray structure of the diol dehydratase–AdePeCbl complex revealed that there are a cobalamin-binding site and an adenine-binding pocket for AdoCbl [17]. The steric strain model is based on a modeling study which showed that super- position of both cobalamin moiety and adenine ring of AdoCbl on those of the enzyme-bound AdePeCbl is not possible without cleavage of the Co-C bond. The adenine ring of the coenzyme would be accommodated to the adenine-binding pocket in order to obtain the maximal binding energy. Supposing AdoCbl is tightly bound by the enzyme at both the cobalamin moiety and the adenine ring, marked distortions, namely both Fig. 4. Photostability of the Co-C bond of diol dehydratase-bound homoadenosylcobalamins. (A, B) Free AdoMeCbl (A) or AdoEtCbl (B) (3.8 l M)in35mM potassium phosphate buffer (pH 8.0) contain- ing 1 M propane-1,2-diol (solid lines). Spectra of the photolyzed ana- logues were also taken after illumination for 1 min with a 250-W tungsten light bulk at a distance of 15 cm (broken lines). (C, D) Apodiol dehydratase (100 unitsÆmL )1 ,6.4lM) was incubated with 3.5 l M of AdoMeCbl (C) or AdoEtCbl (D) in 35 mM potassium phos- phate buffer (pH 8.0) containing 1 M propane-1,2-diol, in a volume of 1.0 mL. Spectra were taken at 5-min of incubation (solid lines). The mixtures were then illuminated for 1 min under the same con- ditions as described in (A) and (B), and spectra were taken (broken lines). M. Fukuoka et al. Coenzymic functions of homoadenosylcobalamins FEBS Journal 272 (2005) 4787–4796 ª 2005 FEBS 4791 angular strains and tensile force, are produced that inevitably break the Co-C bond. We believe that these are molecular entities of the activation of the coen- zyme’s Co-C bond by apoenzyme. Pratt speculated about a similar idea from a chemical viewpoint [30]. The crystal structure of the homolysis fragment cob(II)alamin also suggested that a major contribution to Co-C bond activation in AdoCbl-dependent enzymes would come about ‘by way of apoenzyme (and substrate) induced separation of the homolysis fragments, made possible by strong binding of both separated fragments to the protein’ [31]. A steric strain model has been proposed by Kratky and coworkers as well with glutamate mutase [18]. As shown in Fig. 5, if the group inserted between C5¢ and Co is a methylene, the steric strains induced upon the binding to apoprotein would be largely but not completely relieved. Thus, it would be expected that only a small fraction of the enzyme-bound Ado- MeCbl undergoes the Co-C bond cleavage. As a result, only a trace of coenzymic activity was observed with this analogue. Such speculation is reasonable, because the modeling study revealed that the Co-C distance is elongated to at least 3.3 A ˚ and the Co-C bond leans toward N22 of pyrrole ring B at a C5¢-Co-N22 bond angle of 52° [17]. If a dimethylene group is inserted between C5¢ and Co, the steric strains could be com- pletely relieved. Hence, no cleavage of the Co-C and thus no activity can be expected. This is the case with AdePeCbl as well; that is, the steric strains become invalid with this analogue because of the flexibility of pentamethylene group [27,32]. Thus, all the data repor- ted here are consistent with the steric strain model that we have proposed. To elucidate the mechanism of enzymatic activation (labilization) of the coenzyme Co-C bond, the struc- ture–function relationship of AdoCbl and the role of each structural component of the coenzyme were extensively studied using various coenzyme analogues [24,29,33–35]. Coenzyme analogues, in which one of the structural components of the coenzyme is substi- tuted by a closely related group, were synthesized and examined for coenzymic activity and binding affinity for the enzyme. It was demonstrated that the adenine ring and the ribosyl moiety of the adenosyl group are required for tight binding to the apoenzyme and for transmitting strains to the Co-C bond, respectively, both being indispensable for the Co-C bond activation (catalytic radical formation) and coenzymic function [27,36–38]. Adeninylethylcobalamin undergoes Co-C bond cleavage upon binding of apodiol dehydratase, whereas adeninylpropylcobalamin and other longer chain homologues do not [32]. These lines of evidence suggest the presence of adenine-binding site in the apo- enzyme and that the ‘adenine-attracting effect’ of apo- enzyme is a major element that weakens the Co-C bond. However, the specificity for the adenosyl group is slightly different among AdoCbl-dependent enzymes. Glycerol dehydratase shows similar specificity for the adenosyl group [39–42]. In ribonucleotide reductase Fig. 5. Postulated models of the Co-C bond activation of AdoCbl and its homologues upon binding to apoenzyme. Ade, 9-adeni- nyl; [Co], cobalamin. The regions shown by oblique lines and shadows indicate the adenine-binding pocket and the cobalamin- binding site of enzyme, respectively. Coenzymic functions of homoadenosylcobalamins M. Fukuoka et al. 4792 FEBS Journal 272 (2005) 4787–4796 ª 2005 FEBS [24,29,35], the interaction at N3 of adenine is essential but interaction at 6-NH 2 is less important than those in diol dehydratase. Ribonucleotide reductase shows more strict specificity for the ribose moiety than diol dehydratase. For Aristeromycylcobalamin (AdoCbl analogue in which the ribosyl oxygen atom is replaced by -CH 2 -) is 36–44% and 38% as active as AdoCbl in diol dehydratase [27,43,44] and glycerol dehydratase [44], respectively, but it serves as a strong competitive inhibitor for ethanolamine ammonia-lyase [44] and ribonucleotide reductase [29] and a weak competitive inhibitor for methylmalonyl-CoA mutase [44]. 2¢-De- oxyAdoCbl is 31%, 17%, and 5–13% as active as Ado- Cbl for diol dehydratase [38,45], glycerol dehydratase [41], and ribonucleotide reductase [29,35], respectively, but shows only 1–2% activity for methylmalonyl-CoA mutase [46] or no activity for glutamate mutase [45]. Two series of AdoCbl analogues are of special interest. [x-(Adeninyl)alkyl]cobalamins [24,27,29,32,47,48] were useful to know the limit of the distance of Co and N9 of adenine to keep a stable Co-C bond, that is, the Co-C bond is cleaved with adeninylethylcobalamin but not with adeninylpropylcobalamin or its homologues by the binding to diol dehydratase [32]. Among [x-(adenosin-5¢-O-yl)alkyl]cobalamins that mimic the posthomolysis intermediate state of AdoCbl [28], C5 and C6 analogues showed the strongest inhibition for diol and glycerol dehydratases [49] and methylmalonyl- CoA mutase [50], respectively. These analogues were shown to be useful to get information about the dis- tance between Co and C5¢ after homolysis. Recently, the mechanism-based inactivation of diol dehydratase by 3¢,4¢-anhydroadenosine has been reported [51]. The evidence for the hypothetical adenine-binding site was first obtained by biochemical binding experi- ments [52] and then by X-ray crystallographic analysis [17]. It should be noted that the three-dimensional structure of the adenine-binding pocket can reasonably explain the requirements of the structural components of AdoCbl for binding and catalysis, but can not pre- dict the extents of their contributions to the enzyme catalysis. The structural and biochemical studies are complementary to each other. In this paper, we report the coenzymic functions of homoadenosylcobalamins for diol dehydratase and ethanolamine ammonia-lyase. It was shown that these analogues would be useful for obtaining information about the distance between Co and C5¢, which is critical for Co-C bond cleavage. In order to conclude whether these coenzyme analogues are useful probes for exploring the active sites of AdoCbl-dependent enzymes whose structures have not yet been solved, we have to await further investigation with other enzymes whose distance between Co(II) of cob(II)alamin and substrate radicals are closer than that of diol dehydratase. Experimental procedures Materials Partial structures of the coenzyme analogues used in this study are illustrated in Fig. 1B. Crystalline AdoCbl was a gift from Eisai Co. Ltd. (Tokyo, Japan). AdoMeCbl and AdoEtCbl were prepared as described before by Gscho ¨ sser et al. [25], and AdePeCbl as described by Hogenkamp [26]. All other chemicals were reagent-grade commercial prod- ucts and were used without further purification. Apoenzymes of recombinant Klebsiella oxytoca diol dehydratase and Escherichia coli ethanolamine ammonia- lyase were purified to homogeneity from E. coli JM109 cells harboring expression plasmids pUSI2E(DD) [53,54] and pUSI2ENd(EAL) (K. Akita and T. Toraya, to be pub- lished), respectively. Enzyme and protein assays Activities of diol dehydratase and ethanolamine ammonia- lyase were determined by the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method [27]. The reaction mixture contained an appropriate amount of apoenzyme, 15 lm AdoCbl, 0.1 m propane-1,2-diol or ethanolamine, 50 mm KCl, and 30 mm potassium phosphate buffer (pH 8.0), in a total volume of 1.0 mL. After incubation at 37 °C for 10 min, reactions were terminated by adding 1 mL of 0.1 m potassium citrate buffer (pH 3.6). MBTH hydrochlo- ride was then added to a final concentration of 0.9 mm, and the mixtures were incubated again at 37 °C for 15 min. The concentration of aldehydes formed was determined by measuring the absorbance at 305 nm. One unit is defined as the amount of enzyme activity that catalyzes the forma- tion of 1 lmol of propionaldehyde or acetaldehyde per minute at 37 °C under standard assay conditions. Apparent K i values for coenzyme analogues were obtained by the double reciprocal plots at a fixed concentration (1 lm) of an analogue and varied concentrations (0–10 lm)of AdoCbl. The alcohol dehydrogenase-NADH coupled assay method [55] was also used for the assays of both diol dehy- dratase and ethanolamine ammonia-lyase. The reaction mixture contained an appropriate amount of apoenzyme, 10 lm AdoCbl or its analogue, 0.1 m propane-1,2-diol or ethanolamine, 120 lg of yeast alcohol dehydrogenase, 0.4 mm NADH, and 30 mm potassium phosphate buffer (pH 8.0), in a total volume of 1.0 mL. Reactions were initi- ated by adding AdoCbl or its analogue, and a change of the absorbance at 340 nm was recorded. k inact was calcula- ted from a change in the slope of a tangent to the time course curve of the reaction thus obtained. M. Fukuoka et al. Coenzymic functions of homoadenosylcobalamins FEBS Journal 272 (2005) 4787–4796 ª 2005 FEBS 4793 The protein concentration of purified preparations of the enzymes was determined by measuring the absorbance at 280 nm. The molar absorption coefficient at 280 nm (e M,280 ) calculated by the method of Gill and von Hippel [56] from the deduced amino acid compositions and subunit structures were 120 500 and 302 400 m )1 Æcm )1 for diol dehydratase and ethanolamine ammonia-lyase, respectively. Based on the predicted molecular masses, e 1%,280 was calcu- lated to be 5.81 and 6.21 for the former and latter enzymes, respectively [57]. Other analytical procedures The concentrations of organocobalamins were determined spectrophotometrically after converting them to a dicyano form by photolysis in the presence of 0.1 m KCN, using e 367 ¼ 30.4 · 10 3 m )1 Æcm )1 for dicyanocobalamin [58]. Acknowledgements This work was supported in part by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and from the Japan Society for Promotion of Science (13125205 and 13480195 to TT) and by the Austrian Science Foundation (FWF P-13595 to BK). We thank Ms. Yukiko Kurimoto for assistance in manuscript preparation. References 1 Toraya T (2003) Radical catalysis in coenzyme B 12 - dependent isomerization (eliminating) reactions. Chem Rev 103, 2095–2127. 2 Banerjee R (2003) Radical carbon skeleton rearrange- ments: catalysis by coenzyme B 12 -dependent mutases. Chem Rev 103, 2083–2094. 3 Dolphin D, ed (1982) B 12 , Vol. 2 . 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