Chemistry Reference
In-Depth Information
2002; Thauer et al., 2008
). MCR converts methyl-CoM (methyl-SCoM) and N
7
-mercaptoheptanoylthreonine
phosphate (CoBSH) to methane and the CoB-SS-CoM heterodisulfide (
Table 15.1
,
Reaction 6), with a turnover
number of
10
5
m
1
s
1
. The structure of the enzyme from M.
thermoautotrophicum has been determined at high resolution in two states with substrate bound. Ni is present in
the enzyme in a corrinoid cofactor designated F
430
on account of its absorption maximum at 430 nm (
Figure 15.6
)
.
The enzyme is a heterotrimer with two active sites, each with a Ni-containing tetrapyrrole. The cofactor F
430
,
which is active in the Ni(I) state, is non-covalently, but tightly bound, deeply buried in the protein, but connected
to the surface by a 30
˚
long channel through which the substrates enter. The reaction involves the substrates,
methyl-S-coenzyme M (CH
3
-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate, or coenzyme B (CoB-
SH), which are converted into methane and the heterodisulfide CoB-S-S-CoM. Two mechanisms have been
proposed, one involving an organometallic methyl-Ni intermediate and the other a methyl radical (
Figure 15.6
).
100 s
1
and a k
cat
/K
m
(methyl-SCoM) of
1
w
w
COBALAMINE AND COBALT PROTEINS
Vitamin B
12
, identified as the antipernicious anemia factor in 1925, is a tetrapyrrole cofactor in which the central
hexacoordinate cobalt atom is coordinated by four equatorial nitrogen ligands donated by the pyrroles of the corrin
ring (
Figure 15.7
). The fifth Co ligand is a nitrogen atom from a 5,6-dimethylbenzimidazole nucleotide (Dmb)
covalently linked to the corrin D ring. The sixth ligand in vitamin B
12
is
CN. In the coenzyme B
12
(AdoCbl) this
ligand is 5
0
-deoxyadenosine, while in the other biologically active alkylcobalamine (MeCbl), it is a methyl group.
This sixth ligand is unusual in that it forms a C
e
metal bonds are rare in biology. The free
cofactor can exist in the base-on or base-off conformations (
Figure 15.8
), with the Dmb-on form predominant at
physiological pH. In some B
12
-dependent enzymes, an active site His residue replaces the dimethylbenzimidazole
(the so-called His-on form). In the corrinoid iron
e
Co bond
e
carbon
e
sulfur protein (CFeSP involved in the CODH/ACS system
described earlier), the cofactor is in the Dmb-off conformation and a protein ligand does not appear to occupy the
lower axial position. The reactive C-Co bond participates in all three classes of enzymes which use cobalamine
cofactors, namely the adenosylcobalamine-dependent isomerases, the methylcobalamine-dependent methyl-
transferases and the reductive dehalogenases. We will discuss the first two classes in greater detail here in addition
to a number of noncorrin-cobalt-containing enzymes.
e
B
12
-DEPENDENT ISOMERASES
Isomerases are the largest subfamily of B
12
-dependent enzymes found in bacteria, which play important roles in
fermentation pathways. The only exception is methylmalonyl-CoA mutase, an enzyme required for the metabolism
of propionyl-CoA in man as well as in bacteria. The general reaction mechanism (
Figure 15.9
) for AdoCbl-
dependent isomerases involves homolytic formation of a 5
0
-deoxyadenosyl radical (step 1) followed by H
abstraction to generate a substrate radical (step 2). Once the substrate radical has been formed, it can undergo 1,2
rearrangement (step 3) to generate the product radical. Hydrogen abstraction will then result in the product and the
5
0
-deoxyadenosyl radical (step 4), which can revert to the initial B
12
coenzyme (step 5). As pointed out in Chapter
13, some microorganisms, such as Lactobacillii,haveB
12
-dependent Class II ribonucleotide reductases, where
a thiyl radical is generated rather than a substrate radical by the deoxyadenosyl radical. The thiyl radical is common
to both the diiron-tyrosyl radical-dependent and the B
12
-dependent ribonucleotide reductases, and is responsible
in turn for generating the substrate radical. It is interesting to point out that unlike the other B
12
-dependent
isomerases, the B
12
-dependent Class II ribonucleotide reductase has a different fold for binding B
12
, which is
similar to the corresponding structural elements used in the Class I diiron-tyrosyl ribonucleotide reductases.
The Co
e
carbon bond in AdoCbl is stable in water, but is inherently labile, with a bond dissociation energy of
35 kcal mol
1
. This instability is exploited by the AdoCbl-dependent isomerases to effect radical-
based rearrangements which, as pointed out above, are initiated by homolytic cleavage of the Co-carbon bond.
In the absence of substrate, the homolysis products are not observed, yet in their presence the homolytic cleavage
around 30
e
