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BY JONATHAN D. BRODIE
DEPARTMENT OF BIOCHEMISTRY, STATE UNIVERSITY OF NEW YORK AT BUFFALO
Since the original isolation of deoxyadenosyl-B12, a number of unusual enzymatic transformations catalyzed by this coenzyme have been delineated.' In
general, these reactions may be viewed as a double 1,2 shift in which one of the
migrating groups is hydrogen.2
The central role of the carbon-cobalt bond in the catalytic function of B12
coenzymes has been revealed in the last few years in the laboratories of Abeles3
and Arigoni,4 who used the deoxyadenosyl-B12-dependent propanediol dehydrase
system from A. aerogenes that catalyzes the reaction:
OH
CH37---CH20H~z:CH3CH2--CH(OH)2i--CH,3CH2CHO.
H
The labeling data have clearly established the mechanism to be the abstraction of
hydride by the 5' carbon of the coenzyme followed by hydroxyl migration and the
addition of hydride from the coenzyme to the substrate. 4
The similar 1,2 shift catalyzed by methylmalonyl CoA mutase,
COOH
CH3CH=HOOC-CH2-CH2COSCoA,
COSCoA
cannot be rationalized by an analogous hydride mechanism, since this would
require attack of a primary carbonium ion on the carbonyl carbon of the thiolester.
The hydrogen transfer mechanisms are probably disparate, although the incorporation of hydrogen from the substrate to the 5' carbon of the adenosyl moiety of
the coenzyme appears to be identical to that observed in propanediol dehydrase.'
The cleavage of the carbon-cobalt bond appears to be an intrinsic step in all
known B,2-dependent reactions.2 Accordingly, we wish to report on the nature
of this carbon-cobalt bond as delineated by ligand binding studies and the spatial
relationship of the substituents of the d2 orbital to the corrinoid system.
461
BIOCHEMISTRY: J. D. BRODIE
462
PRoc. N. A. S.
Materials and Methods.-Vitamin B12 was purchased from Pierce Chemical Co. A
series of alkyl halides and substituted amines were products of Distillation Products
Industries. Aquocobinamide was prepared by the cerous ion-catalyzed hydrolysis of
aquocobalamin (B12a) as previously described.6 Alkyl cobinamides were prepared by
borohydride reduction of aquocobinamide to Co'-cobinamide followed by addition of the
appropriate alkyl bromide or iodide in the dark, and purified by chromatography on
Sephadex G-10 and CM-cellulose.
Ligand binding studies were performed at room temperature (230) in the dark, and
spectra were recorded with a Cary 14 recording spectrophotometer.
Results.-Properties of secondary alkyl cobinamides: Cyclohexyl cobinamide
and sec-butyl cobinamide gave essentially identical spectra. Figure 1 shows the
spectrum of cyclohexyl cobinamide before and after photolysis. The photolysis
1.0
0.5,
O.D.
2501
3501
___
4501
5501
650
BIOCHEMISTRY: J. D. BRODIE
O.D.
Ir
250
463
350
450
I
450
550
650
O.D.
Iftf
t hw
in the d6 position profoundly alters the spectrum to yield a spectrum very close
to that of methyl cobalamin. Titration of methyl cobinamide with NH40H and
pyridine gives the results shown in Figures 4 and 5. It can be seen that the
nature of the nitrogenous ligand has very minor effects on the spectra, with
differences noted only in the region near 340 m. MIethylamine and ethylamine
complexes form at concentrations intermediate between ammonia and pyridine.
Piperidine, although an excellent nucleophile, is a very poor ligand-former,
presumably due to steric hindrance. We have not been able to detect interaction
of any amine with cyclohexyl cobinamide, even upon raising the temperature to
60.
464
PROC. N. A. S.
BIOCHEMISTRY: J. D. BRODIE
1.0-
0.5-
0.0.
250
350
450
550
650
05Ak
250
350
450
550
65(
mL
FIG. 5.-Titration of methyl cobinamide with pyridine. Methyl cobinamide (1.7 X 10' M) was titrated with pyridine to a final concentration of
0.66 M base. The ligand concentration for 50% change in absorbance at 305,
340, 460, and 515 my was determined to be 0.11 M.
BIOCHEMISTRY: J. D. BRODIE
465
CN
Co0 CH3I
C-h
(CN
QCN
INYCo
N-CH3
CN
Co
N-CH3
CN
CL
NaBH
0
C N-CH3
A control experiment showed that the phosphate ester linking the nucleoside
to the C ring of the corrin was not hydrolyzed by the quaternizing conditions.
4;66
BIOCHEMISTRY: J. D. BRODIE
PRoc. N. A. S.
d5
Co
d6
kd
II
alkylation of aquocobalamin, in which the d6 position is coordinated with nitrogen-7 of the dimethylbenzimidazole, could not be effected. Although it might
be argued that the secondary alkyl substituent is destabilized by induction, it
is probable that in the case of the cobalamins, the trans inductive effects are
minimal when compared with steric factors.8
The experimental results support this view. Thus, the Col cobinamide can be
alkylated by a bulky substituent because of the absence of a strong ligand in the
d6 position, which allows the cobalt to remain above the plane of the corrin
system, which hinders the binding of nitrogenous ligands. Conversely, the
presence of a strong ligand (dimethylbenzimidazole) in the Co'-cobalamin
series tends to force the cobalt into the plane of the corrin, which precludes
the formation of a carbon-cobalt bond with a bulky alkyl group. This is consistent with the alkaline cyanide decomposition of cyclohexyl cobinamide (which
may be visualized as the addition of a strong ligand to the d6 position that tends to
force the cobalt into the plane of the corrin, with concomitant labilization of the
carbon-cobalt bond caused by steric hindrance) followed by rapid addition of
cyanide to give the observed dicyanocobinamide, with no evidence of a stable
monocyano cyclohexyl derivative.' More direct support for this view may be
found by examination of the synthesis of the cyclohexyl-"cobalamin" that is
formed only after quaternization of the nitrogenous ligand.
An understanding of the enzymatic behavior of the B,2 coenzymes may be
provided by the preceding considerations in which a hydride transfer mechanism
(operative for propanediol dehydrase) would be facilitated by a carbon-cobalt
cleavage into a Co1-R9 species that would be stabilized by the absence of an
electron donor in the d6 position. This would suggest that in carbonium ion
transfers, as in methionine biosynthesis'0 or hydride abstractions (cf. ref. 33 cited
in ref. 2), the enzyme binds the coenzyme in a manner in which the dimethylbenzimidazole is not coordinated with the cobalt. On the other hand, a proton
abstraction reaction, which may be used to explain the isomerization of methylmalonyl CoA to succinyl CoA, would be visualized as involving a heterolytic
carbon-cobalt cleavage to form a Co,,'-Re pair in which the dimethylbenzimidazole coordinated to the cobalt would stabilize the intermediate CoIII species.
Thus, it appears likely that the key transition in all coenzyme-Bn2 catalyzed
enzymatic reactions may be effected merely by altering the distance of the
cobalt-nitrogen bond, which in turn changes the symmetry of the cobalt and the
polarization of the carbon-cobalt bond. We shall test this hypothesis by circular
BIOCHEMISTRY: J. D. BRODIE
467
dichroism and magnetic resonance studies with model compounds and appropriate enzymatic experiments.
The author gratefully acknowledges Mr. Melvin Spadford for his excellent technical
assistance, Drs. Curtis Hare, R. G. Wilkens, and R. M. McLean for helpful discussions,
and Dr. W. B. Elliott for the use of his Cary 14 spectrophotometer (provided by USPHS
grant no. GM-06241).
* This work was supported by grant no. AM-10479 from the National Institutes of Health.
lBaker, H. A., H. Weissbach, and R. D. Smyth, these PROCEEDINGS, 44, 1093 (1958).
2 Hogenkamp, H. P. C., Ann. Rev. Biochem., 37, 225 (1968).
3 Frey, P. A., M. K. Essenberg, and R. H. Abeles, J. Biol. Chem., 242, 5369 (1967).
4 R6tey, J., A. Umani-Ronchi, J. Seibl, and D. Arigoni, Experientia, 22, 502 (1966).
5 Cardinale, G. J., and R. H. Abeles, Biochim. Biophys. Ada, 132, 517 (1967).
6 Friedrich, W., and K. Bernhauer, Chem. Ber., 89, 2507 (1956).
7 Ibid., p. 2030.
8 Schrauzer, G. N., and R. J. Windgassen, J. Am. Chem. Soc., 89, 1999 (1967).
9 Hogenkamp, H. P. C., J. E. Rush, and C. A. Swenson, J. Biol. Chem., 240, 3641 (1965).
10 The experimental data obtained with methionine synthetase are essentially considered in
this light by Hogenkamp.2