From the Department of Biochemistry, University of
Nebraska, Lincoln, Nebraska 68588-0664 and
§ Department of Bacteriology, University of Wisconsin,
Madison, Wisconsin 53706-1567
Received for publication, July 31, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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Methylmalonyl-CoA mutase is an
5'-adenosylcobalamin (AdoCbl)-dependent enzyme that
catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The
crystal structure of this protein revealed that binding of the cofactor
is accompanied by a significant conformational change in which
dimethylbenzimidazole, the lower axial ligand to cobalt in solution, is
replaced by His610 donated by the active site.
The role of the lower axial ligand in the trillion-fold labilization of
the upper axial cobalt-carbon bond has been the subject of enduring
debate in the model inorganic literature. In this study, we have used a
cofactor analog, 5'deoxyadenosylcobinamide GDP (AdoCbi-GDP), which
reconstitutes the enzyme in a "histidine-off" form and which allows
us to evaluate the contribution of the lower axial ligand to catalysis.
The kcat for the enzyme in the presence of
AdoCbi-GDP is reduced by a factor of 4 compared with the native cofactor AdoCbl. The overall deuterium isotope effect in the presence of AdoCbi-GDP (DV = 7.2 ± 0.8) is
comparable with that observed in the presence of AdoCbl (5.0 ± 0.6) and indicates that the hydrogen transfer steps in this reaction
are not significantly affected by the change in coordination state of
the bound cofactor. These surprising results are in marked contrast to
the effects ascribed to the corresponding lower axial histidine ligands
in the cobalamin-dependent enzymes glutamate mutase and
methionine synthase.
Coenzyme B12- or
AdoCbl1-dependent
enzymes catalyze a wide variety of isomerization reactions in which a
migrating group and a hydrogen atom on vicinal carbons exchange
positions. A common function of the cofactor in these reactions is to
serve as a dormant source of radicals that is activated on substrate
binding by homolysis of the organometallic Co-C bond (1-3). The
uncatalyzed rate for the cleavage of the Co-C bond in the cofactor
free in solution is 3.8 × 10 In solution and at physiological pH, AdoCbl is six-coordinate,
and the lower axial ligand is the bulky and weakly basic intramolecular base dimethylbenzimidazole (9). The potential role of the lower axial
ligand in labilizing the upper axial Co-C bond has been the focus of
much discussion. A popular hypothesis to explain the observed rate
enhancement invokes the role of conformational distortion of the corrin
macrocycle (e.g. Refs. 10-14). According to the
"mechanochemical" mechanism for labilization of the Co-C bond, an
upward flexing of the corrin ring would lead to steric crowding on the
On the basis of these model studies, a bulky and weakly basic ligand
such as dimethylbenzimidazole would appear to be well suited for
AdoCbl-dependent enzymes capable of effecting spectacular enhancements of the Co-C bond homolysis rate. It therefore came as a
surprise when spectroscopic investigations (20, 21) and crystal
structures (22, 23) revealed that the cofactor was bound in a
"dimethylbenzimidazole-off" conformation in some
AdoCbl-dependent enzymes. This ligand switch was first
reported in B12-dependent methyltransferases
that catalyze heterolytic cleavage of the Co-C bond (24, 25). In all
these enzymes, a histidine residue donated by the protein replaces the
intramolecular ligand, dimethylbenzimidazole.
In methylmalonyl-CoA mutase, His610 is coordinated to the
cobalt and is part of a hydrogen-bonding triad involving
Asp608 and Lys604 (22). We have previously
shown that a major role for the dimethylbenzimidazole-containing tail
in methylmalonyl-CoA mutase is to organize the active site for
catalysis (26). Thus, the cofactor analogs AdoCbi and
5'-deoxyadenosylcobinamide phosphate methylester, lacking the
dimethylbenzimidazole moiety, bind tightly to the enzyme but do not
support catalysis. On the basis of comparisons of the crystal
structures of holomethylmalonyl-CoA mutase (22) and of the
B12-binding domain of glutamate mutase lacking the cofactor
(27), we had postulated that the role of the bulky base was to organize
a disordered loop in the apoenzyme to a helix found in the holoenzyme
(26). In this study, we have used an AdoCbl analog, AdoCbi-GDP (Fig.
1), in which the terminal base on the nucleotide tail,
dimethylbenzimidazole, is replaced by GDP. This derivative supports
catalysis with only slightly reduced efficiency but, surprisingly, does
so from a "histidine-off" conformation. These results suggest that
the lower axial ligand His610 apparently plays a minor role
in the reaction catalyzed by methylmalonyl-CoA mutase.
Materials--
AdoCbl, GDP, and methylmalonlyl-CoA were
purchased from Sigma. Radioactive
[14C]CH3-malonyl-CoA (56.4 Ci/mol) was
purchased from New England Nuclear. Thiokinase was purchased from Roche
Molecular Biochemicals. [CD3]-Methylmalonyl-CoA (28) and
AdoCbi-GDP (29) were synthesized as described previously. Titanium
citrate was prepared as described previously (30). All other chemicals
were reagent-grade commercial products and were used without further purification.
Enzyme Expression and Purification--
The recombinant
expression vector pMEX2/pGP1-2 harboring the
Propionibacterium shermanii genes in
Escherichia coli strain K38 (31) was a gift from Peter
Leadlay (Cambridge University). The enzyme was purified through the
step preceding reconstitution with cofactor as described before
(32).
Enzyme Assays--
The specific activity of the mutase was
determined in the radiolabeled assay at 37 °C as described
previously (33) and was 26 units/mg of protein. 1 unit of activity
catalyzes the formation of 1 µmol of succinyl-CoA/min at 37 °C.
The deuterium isotope effect was measured with
[CD3]methylmalonyl-CoA in the coupled thiokinase assay at
30 °C as described previously (33).
Determination of Equilibrium Binding Constants for AdoCbl and
AdoCbi-GDP by Fluorescence Spectroscopy--
The intrinsic tryptophan
fluorescence emission of methylmalonyl-CoA mutase (monitored at 340 nm)
is quenched on binding of AdoCbi-GDP as also seen with AdoCbl (26).
Fluorescence measurements of equilibrium binding constants were made on
a PerkinElmer LS50 luminescence spectrometer as described previously
(26). The excitation wavelength was 282 nm (slit width, 3 µm), and
emission was observed between 300 and 380 nm (slit width, 3 µm). To
determine the equilibrium dissociation constant for AdoCbi-GDP, 500 µl of 0.25 µM methylmalonyl-CoA mutase in 50 mM potassium phosphate buffer, pH 7.5, was used. Successive
aliquots (2-5 µl) of a 0.1 mM stock AdoCbi-GDP solution
prepared in the same buffer were added to the enzyme solution. After
each addition, the mixture was incubated at 4 °C for 30 min before
measurement of the fluorescence emission. Free AdoCbi-GDP, even at
millimolar concentrations, does not exhibit fluorescence emission
between 300 and 600 nm on excitation at 282 nm. As an additional
control for nonspecific quenching, the fluorescence of lysozyme was
monitored on addition of millimolar concentrations of AdoCbi-GDP. No
change in the fluorescence emission was observed. Binding data were
analyzed as described previously to obtain the respective
Kd values for AdoCbl and AdoCbi-GDP (26).
Equilibrium Binding Constants Measured by UV-visible Absorption
Spectroscopy--
Binding of AdoCbl and AdoCbi-GDP to
methylmalonyl-CoA mutase was followed spectrophotometrically using a
Cary-118 spectrophotometer (Olis Instruments), in which the cuvette
holder was maintained at 4 °C by a thermostatted water circulator.
Methylmalonyl-CoA mutase (18.2 µM) in 150 µl of 50 mM potassium phosphate buffer, pH 7.5, was used as a blank.
Spectra were recorded between 800 and 306 nm after each addition of
AdoCbi-GDP (3-5-µl aliquots prepared in the same buffer), after
incubation at 4 °C for 30 min. The final AdoCbi-GDP concentration
used in these experiments was 43.1 µM. The change in
absorbance at 460 nm at each concentration of AdoCbi-GDP was
obtained by subtracting the spectrum of the same concentration of free
AdoCbi-GDP in 50 mM potassium phosphate buffer, pH 7.5. Data were analyzed as described previously (26). A
Kd of 4.9 µM obtained from the
equilibrium fluorescence measurements was used to generate the
best-fit line to the binding data obtained by absorption spectroscopy.
EPR Spectroscopy--
The EPR spectrum was recorded on a Bruker
ESP 300E spectrometer equipped with an Oxford ITC4 temperature
controller, a model 5430 automatic frequency counter from Hewlett
Packard, and a gauss meter. The specific conditions used for spectral
recording are provided in the figure legends. The holeoenzyme sample
was prepared as follows. Apomethylmalonlyl-CoA mutase (240 µM) in 50 mM potassium phosphate buffer, pH
7.5, was mixed with an equal volume of AdoCbi-GDP (500 µM) in the same buffer. The solution was incubated at
4 °C for 30 min before passing through a G25 Penefsky column to
remove unbound cofactor. The spectrum of the enzyme was recorded. To generate the paramagnetic cob(II)inamide-GDP state of the enzyme, an
anaerobic solution containing holoenzyme (110 µM) and 95 µM titanium citrate was placed in an EPR tube on ice and
exposed for 1 h to a 60-W tungsten lamp placed at a distance of 15 cm, and the sample was subsequently frozen. Titanium citrate was added at a low concentration to scrub out residual oxygen in the solution.
Spectral Properties of Methylmalonlyl-CoA Mutase Reconstituted with
AdoCbi-GDP--
AdoCbi-GDP is an intermediate in the biosynthesis of
AdoCbl (34) and exists in the
"base-off"2 conformation
in solution (Fig. 1). Binding of
AdoCbi-GDP to wild-type mutase results in an increase in absorption
across the entire spectral range (Fig.
2), as has been observed previously for
AdoCbl (26). The spectrum of the bound cofactor retains the 460-nm absorption maximum, indicating that His610 in the active
site is not coordinated to the cobalt in AdoCbi-GDP-reconstituted enzyme (Fig. 1). The increase in absorbance at 460 nm accompanying cofactor binding was plotted as a function of the concentration of free
cofactor at equilibrium (Fig. 2, inset). The free ligand concentration was calculated at each point in the titration curve by
using the Kd obtained from the fluorescence
experiments (4.9 µM) as described below (26). The
excellent fit to the absorbance data using the Kd
value obtained from the fluorescence data confirms that the binding
isotherm obtained by fluorescence spectroscopy reflects specific
binding of the cofactor to the mutase.
Steady-state Kinetic Properties of AdoCbi-GDP-reconstituted
Methylmalonyl-CoA Mutase--
Despite the lower affinity of
methylmalonyl-CoA mutase for AdoCbi-GDP, it is a relatively good
cofactor analog supporting turnover with a kcat
that is only 4-fold lower than the natural cofactor AdoCbl (Table
I). The Km for the
substrate methylmalonyl-CoA is not affected by the cofactor analog
(data not shown). The overall deuterium isotope effect under
steady-state conditions in the presence of AdoCbl
(DV = 5.0 ± 0.6) and AdoCbi-GDP
(DV = 7.2 ± 0.8) are similar to each
other and to the isotope effect (DV = 6.2)
reported previously for wild-type methylmalonyl-CoA mutase in the
presence AdoCbl (35).
Determination of Equilibrium Binding Constants for Cofactors by
Fluorescence Spectroscopy--
Addition of AdoCbl and AdoCbi-GDP to
methylmalonyl-CoA mutase results in a decrease in fluorescence emission
at 340 nm (data not shown) that can be used to determine the
equilibrium dissociation constant (26). The Kd for
AdoCbi-GDP is 4.9 ± 0.3 µM, which is 30-fold higher
than that for AdoCbl (Table I).
Electronic Absorption Spectrum of AdoCbi-GDP-reconstituted
Methylmalonyl-CoA Mutase under Steady-state Turnover
Conditions--
The spectrum of the enzyme monitored under
steady-state turnover conditions reveals the predominance of the
AdoCbi-GDP state (Fig. 3). However, the
spectrum of the enzyme in the presence of substrate is slightly blue
shifted, and the absorption maximum shifts from 460 to 456 nm. Two
isosbestic crossovers are observed at 454 and 496 nm, respectively. In
the presence of the natural cofactor, the steady-state spectrum of the
enzyme comprises of a mixture of AdoCbl (~80%) and cob(II)alamin
(~20%; Ref. 36).
EPR Spectrum of Cob(II)inamide-GDP-reconstituted Methylmalonlyl-CoA
Mutase--
To confirm that the mutase-bound cofactor was in the
base-off conformation, the EPR spectrum of the photolyzed enzyme was recorded (Fig. 4). The axial spectrum is
diagnostic of base-off cob(II)inamide in which the
gII component is split into eight lines centered at g = 2.002 because of hyperfine coupling between the unpaired electron and
cobalt nucleus (I = 7/8). The hyperfine coupling constant is 144 G. The g Clearly, cobalt coordination by the bulky intramolecular base,
dimethylbenzimidazole, is not essential for catalysis in
AdoCbl-dependent isomerases. Thus, in a subfamily of these
enzymes including methylmalonyl-CoA mutase and glutamate mutase, the
nucleotide loop is bound in an extended conformation and is replaced by
a histidine ligand donated by the protein (22, 23). In contrast,
coordination by dimethylbenzimidazole is preserved in the active site
in a second subfamily (39-42). However, regardless of the identity of
the lower axial ligand, its role in catalysis is poorly understood.
In a previous study, we had reported that deletion of the nucleotide
base from the cofactor tail resulted in inactive cofactors that
retained tight binding to methylmalonyl-CoA mutase (26). In this study,
we have examined the binding and catalytic properties of AdoCbi-GDP.
Surprisingly, this analog supports catalysis but binds to the active
site in a histidine-off conformation and thus permits evaluation of the
contribution of the lower axial ligand to the overall reaction.
AdoCbi-GDP differs from AdoCbl in three important structural respects
(Fig. 1). First, the anomeric carbon in GDP is in the AdoCbi-GDP binds to methylmalonyl-CoA mutase with an ~30-fold lower
affinity (Table I). As a cofactor, AdoCbi-GDP is rather efficient, and
the kcat for methylmalonyl-CoA mutase is only
4-fold lower than with AdoCbl. In addition, the overall deuterium
isotope effects on the reactions supported by AdoCbl and AdoCbi-GDP are similar (Table I). Thus, the intrinsic deuterium isotope effect associated with hydrogen transfer from deoxyadenosine to substrate (36,
44) is suppressed to a similar extent in both cases. This is
significant, because the hydrogen transfer step from substrate to
cofactor (and presumably from cofactor to product) is kinetically coupled to the cobalt-carbon bond homolysis step (36). These results
suggest that the absence of histidine coordination does not affect the
coupled homolysis step, because the overall energetics of the reaction,
as indicated by the isotope effects, are similar.
Thus the lower axial ligand His610 apparently plays a minor
role in the reaction catalyzed by methylmalonyl-CoA mutase,
contributing marginally, if at all, to the 0.9 × 1012-fold acceleration of the Co-C homolysis reaction rate
(8). This is significantly lower than the 870-fold enhancement of the homolysis rate constant attributed to N-methylimidazole
coordination to AdoCbi in model studies (45). This could be explained
by significant differences in the free energy profiles of the catalyzed and uncatalyzed reactions (8). The seemingly minor role played by the
lower ligand in the reaction catalyzed by methylmalonyl-CoA mutase is
in apparent contrast to the rather significant changes that have been
reported in the related isomerase glutamate mutase (46) and in the
methylcobalamin-dependent enzyme methionine synthase (47,
48). In all three enzymes, the
DXXHXXG motif is seen (49), in which
the conserved histidine and aspartate residues are involved in a
hydrogen bonding network ("the catalytic triad"; Ref. 25), and the
histidine serves as the lower ligand to cobalt. In glutamate mutase,
both conservative and nonconservative mutations of the coordinating
histidine residue lead to significantly increased Kd
values for the cofactor and are accompanied by an
~103-fold lower kcat. In contrast
to methylmalonyl-CoA mutase, glutamate mutase binds AdoCbl relatively
weakly (Kd = 1.8 µM; Ref. 46).
Therefore, coordination by the lower axial ligand may be more important
for cofactor binding in glutamate mutase than in methylmalonyl-CoA mutase.
In methionine synthase, mutation of the coordinating histidine residue
His759 to glycine results in retention of strong cofactor
binding and an ~105-fold diminution in catalytic activity
(47). The base-off state in this enzyme is associated with switching
the protein conformation from the catalytic to the reductive activation
mode (50). Thus, it is likely that the large catalytic penalty incurred
by the H759G mutation in methionine synthase resulted from the protein being frozen in an inactive conformation rather than from effects on
the chemical steps in the cobalamin-dependent
methyltransfer reaction. This is supported by the observation that the
susceptibility of the H759G mutant to limited proteolysis is similar to
that of inactive wild-type enzyme, suggesting that the enzyme exists in
the conformation that binds the repair protein, flavodoxin (51). The
effects of conservative mutagenesis at His759 have not been reported.
In summary, our studies reveal that although the absence of
dimethylbenzimidazole from its binding pocket is marked by failure of
the cofactor to support catalysis and of His610 to
coordinate to cobalt, the presence of an analog such as GDP results in
a relatively high level of enzyme activity, albeit in the absence of
His610 ligation. An alternative and perhaps more direct
evaluation of the contribution of His610 to catalysis would
be by site-directed mutagenesis of the residue, and these studies are
in progress in our laboratory. The present study reveals that the lower
axial ligand apparently plays a minor role in the rearrangement
reaction catalyzed by methylmalonyl-CoA mutase, and its presence does
not confer a significant catalytic advantage. We speculate that
retention of the cofactor binding mode with the dimethylbenzimidazole
to histidine ligand switch represents an evolutionary vestige in
methylmalonyl-CoA mutase, because histidine ligation is not critical
for cofactor binding or for the catalytic efficiency of the enzyme.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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9/s at
37 °C (4). In contrast, the kcat for most
AdoCbl-dependent enzymes is on the order of
~102/s, leading to a predicted rate enhancement that is
on the order of 1 trillion-fold (5). A member of this class of enzymes
is methylmalonyl-CoA mutase, which catalyzes the 1-2 rearrangement of
methylmalonyl-CoA to succinyl-CoA (for review, see Refs. 6, 7). It is
distinguished by being the only family member that is found in both
bacterial and mammalian organisms. Methylmalonyl-CoA mutase catalyzes a
0.9 × 1012-fold enhancement of the homolysis rate
that corresponds to a lowering of the activation barrier by 17 kcal/mol
at 37 °C (8).
face, thereby weakening the organometallic bond. The influence of
trans-steric and electronic effects exerted by the lower axial ligand
has been examined in a number of model compounds (15-19).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of free and
mutase-bound conformations of (A) AdoCbl and
(B) AdoCbi-GDP. R, deoxyadenosine.
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Fig. 2.
UV-visible spectrum of free and enzyme-bound
AdoCbi-GDP. The dotted line represents the spectrum of
8.9 µM free AdoCbi-GDP in 50 mM potassium
phosphate buffer, pH 7.5, and the solid line is the spectrum
of 8.9 µM AdoCbi-GDP with 30.1 µM
methylmalonyl-CoA mutase in the same buffer. Inset, plot of
A460 nm as a function of free AdoCbi-GDP
concentration at equilibrium. The concentration of free AdoCbi-GDP was
calculated using Equations 2 and 3 from Chowdhury and Banerjee (26) and
a value of Kd for AdoCbi-GDP of 4.9 µM. The solid line represents a fit obtained
with Equation 4 from Chowdhury and Banerjee (26).
Comparison of steady-state kinetic parameters with AdoCbl versus
AdoCbi-GDP
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Fig. 3.
UV-visible spectrum of methylmalonlyl-CoA
mutase reconstituted with AdoCbi-GDP under steady-state turnover
conditions. An anaerobic solution of holoenzyme (16.7 µM) in 50 mM potassium phosphate buffer, pH
7.5 (a), was mixed with 12 mM
(R,S)-methylmalonlyl-CoA, and the spectrum was recorded
(b).
component is broad and poorly resolved, and the
overall spectrum resembles that of the clostridial corrinoid
iron-sulfur protein that also contains a base-off corrin (37). The
absence of histidine ligation to cob(II)inamide-GDP in the mutase
active site is indicated by the following two observations. The
hyperfine coupling constant, which is 144 G, is typical of a base-off
species and notably higher than that expected for a base-on species,
which is of the order of 110 G (38). Second, the high-field signals
appear as singlets rather than triplets, indicating the absence of
superhyperfine coupling between the unpaired electron and an I = 1 nucleus, as has been observed for histidine ligation in
methylmalonyl-CoA mutase (20). Together, these data provide convincing
evidence for the base-off conformation of AdoCbi-GDP bound to the
mutase active site.
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Fig. 4.
X-band EPR spectrum of methylmalonlyl-CoA
mutase containing cob(II)inamide-GDP. An anaerobic solution of
enzyme (101 µM) in 50 mM potassium phosphate,
pH 7.5, containing 95 µM titanium citrate was photolyzed
as described under "Experimental Procedures." The spectrum was
recorded using the following conditions: sweep width, 2000 G;
modulation frequency, 100 KHz; modulation amplitude, 10 G; power, 5 mW;
temperature, 25 K. The resonator frequency was 9.446 GHz. The line
marker is at g = 2.002.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
configuration
(versus
in the dimethylbenzimidazole nucleotide of
AdoCbl). Second, the nucleotide loop has a diphosphate moiety in
AdoCbi-GDP and a monophosphate moiety in AdoCbl. Third, the phosphate
group is at the 5' position of the ribose ring in AdoCbi-GDP and at the
3' position in AdoCbl. When bound to the mutase, AdoCbi-GDP remains in
the base-off conformation, as evidenced by the 460-nm absorption
maximum (Fig. 2) and the EPR spectrum (Fig. 4). Thus, it appears that
although the occupancy of the nucleotide binding pocket is essential
for catalysis, the structure of the nucleotide is not as critical.
Consistent with this conclusion is the report that
deoxyadenosine-(p-cresolyl)cobamide, in which
dimethylbenzimidazole is replaced by p-cresol, supports
methylmalonyl-CoA mutase activity (43). However, because the spectrum
of the enzyme in the presence of this analog was not reported, it is
not known whether His610 is coordinated to the cobalt in
the active site when p-cresol rather than
dimethylbenzimidazole is positioned in the nucleotide pocket.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK45776 (to R. B.) and GM40313 (to J. C. E.-S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Biochemistry, University of Nebraska, N133 Beadle Center, Lincoln, NE 68588-0664. Tel.: 402-472-2941; Fax: 402-472-7842; E-mail: rbanerjee1@unl.edu.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M006842200
2 "Base-off" and "base-on" are used generically to refer to cobalamin conformations in which the lower axial ligand is absent or present, rather than specifically to the state of dimethylbenzimidazole ligation.
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ABBREVIATIONS |
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The abbreviations used are: AdoCbl, 5'-deoxyadenosylcobalamin or coenzyme B12; EPR, electron paramagnetic resonance; AdoCbi, 5'-deoxyadenosylcobinamide; AdoCbi-GDP, 5'-deoxyadenosylcobinamide guanosine diphosphate.
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