active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase Masaki Fukuoka1, Yuka Nakanishi1, Renate B.. The interactions of the coenzyme’s adenine moiety with
Trang 1active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase
Masaki Fukuoka1, Yuka Nakanishi1, Renate B Hannak2, Bernhard Kra¨utler2and Tetsuo Toraya1
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 kcat and kcat⁄ Km values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regu-lar coenzyme with diol dehydratase, respectively The kcat⁄ kinact value showed that the holoenzyme with this analogue becomes inactivated on average after about 3000 catalytic turnovers, indicating that the probability
of inactivation during catalysis is almost 500 times higher than that for the regular holoenzyme The kcat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonia-lyase, as judged from the initial velocity, but the holoenzyme with this 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 B12; AdoEtCbl, adenosylethylcobalamin; AdoMeCbl, adenosylmethylcobalamin or homocoenzyme
B 12 ; AdePeCbl, adeninylpentylcobalamin.
Trang 2dehydratase [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 moimoi-ety 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.
Trang 3diol 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
kcat value was about 0.27% that for AdoCbl, and the
rate of mechanism-based inactivation (kinact) with this
analogue was as slow as that with the regular coenzyme
As judged from the kcat⁄ kinact 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 (kcat⁄ Km) 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
kcatb
k inactb (min)1)
k cat ⁄ k inactc
· 10)4
K mc (l M )
kcat⁄ K m
· 10)6 ( M )1Æs)1)
K ic (l M )
kcatb
k inactb (min)1)
k cat ⁄ k inact
· 10)4
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.dFrom [27].eFrom the initial velocity.
Trang 4In 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 (Ki) This fact
indicates that they can not serve as coenzymes
although they are bound tightly to the apoenzyme
The observation that the Ki 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 (kcat) of this analogue is about 0.13% that of AdoCbl, as judged from the initial velocity The rate of mechan-ism-based inactivation (kinact) with this analogue was five times faster than that with the regular coenzyme The kcat⁄ kinact 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 l M ) (B) AdoEtCbl (3.8 l M ) (C), or AdePeCbl (3.8 l M ) (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, 6.4 l M ) was incubated with AdoCbl (3.5 l M ) (E) AdoMeCbl (3.8 l M ) (F), AdoEtCbl (3.8 l M ) (G), or AdePeCbl (3.8 l M ) (H) in 35 m M 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 lL of 1 M potassium phosphate buf-fer (pH 8.0) and 70 lL of 5 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 l M ) was incubated with AdoCbl (3.5 l M ) (I), AdoMeCbl (3.8 l M ) (J), AdoEtCbl (3.8 l M ) (K), or AdePeCbl (3.8 l M ) (L) in 31 m M potassium phosphate buffer (pH 8.0) containing 0.2 M ethanolamine and 5 m M 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.
Trang 5analogues 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 ) in 35 m M 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, 6.4 l M ) was incubated with
3.5 l M of AdoMeCbl (C) or AdoEtCbl (D) in 35 m M 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).
Trang 6angular 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 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.
Trang 7[24,29,35], the interaction at N3 of adenine is essential
but interaction at 6-NH2 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 -CH2-) 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 37C 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 37C 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 37C under standard assay conditions Apparent
Ki 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 kinactwas calcula-ted from a change in the slope of a tangent to the time course curve of the reaction thus obtained
Trang 8The protein concentration of purified preparations of the
enzymes was determined by measuring the absorbance at
280 nm The molar absorption coefficient at 280 nm
(eM,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)1Æcm)1 for diol
dehydratase and ethanolamine ammonia-lyase, respectively
Based on the predicted molecular masses, e1%,280was
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
e367¼ 30.4 · 103m)1Æcm)1for 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
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