Metabolism of pentacarboxylate porphyrinogens by highly purified human coproporphyrinogen oxidase: Further evidence for the existence of an abnormal pathway for heme biosynthesis

Article abstract of DOI:10.1016/j.bmc.2005.06.051

An abnormal series of porphyrin tetracarboxylic acids known as the isocoproporphyrins, are commonly excreted by patients suffering from the disease porphyria cutanea tarda (PCT). These porphyrins appear to arise by bacterial degradation of dehydroisocoproporphyrinogen that is generated by the premature metabolism of the normal pentacarboxylate intermediate (5dab) by coproporphyrinogen oxidase (copro'gen oxidase). This porphyrinogen can be further metabolized by uroporphyrinogen decarboxylase to give harderoporphyrinogen, one of the usual intermediates in heme biosynthesis. Therefore, it is possible that some of the heme formed under abnormal conditions may originate from the 'isocopro-type' porphyrinogen intermediate. In order to investigate the feasibility of alternative pathways for heme biosynthesis, the four type III pentacarboxylate isomeric porphyrinogens were incubated with purified, cloned human copro'gen oxidase at 37°C with various substrate concentrations under initial velocity conditions. Of the four isomers, only 5dab was a substrate for copro'gen oxidase and this gave dehydroisocoproporphyrin. The structure of the related porphyrin tetramethyl ester was confirmed by proton NMR spectroscopy and mass spectrometry. The K_m value for proto'gen-IX formation from copro'gen, an indicator of molecular recognition, was similar to the K_m value for monovinyl product formation with 5dab, although copro'gen-III has an approximately twofold higher K_cat value. Although 5dab is a slightly poorer substrate than copro'gen-III, these results support the hypothesis that an abnormal route for heme biosynthesis is possible in humans suffering from PCT or related syndromes such as hexachlorobenzene poisoning.

Full text of DOI:10.1016/j.bmc.2005.06.051

Bioorganic & Medicinal Chemistry 13 (2005) 6244–6251
Metabolism of pentacarboxylate porphyrinogens by highly
purified human coproporphyrinogen oxidase: Further evidence q
for the existence of an abnormal pathway for heme biosynthesis
Christopher L. Cooper, Christian M. Stob, Marjorie A. Jones and Timothy D. Lash*
*
Department of Chemistry, Illinois State University, Normal, IL 61790-4160, USA
Received 3 June 2005; revised 23 June 2005; accepted 23 June 2005
Available online 3 August 2005
Abstract—An abnormal series of porphyrin tetracarboxylic acids known as the isocoproporphyrins, are commonly excreted by
patients suffering from the disease porphyria cutanea tarda (PCT). These porphyrins appear to arise by bacterial degradation of
dehydroisocoproporphyrinogen that is generated by the premature metabolism of the normal pentacarboxylate intermediate
(
5dab) by coproporphyrinogen oxidase (coproꢀgen oxidase). This porphyrinogen can be further metabolized by uroporphyrinogen
decarboxylase to give harderoporphyrinogen, one of the usual intermediates in heme biosynthesis. Therefore, it is possible that some
of the heme formed under abnormal conditions may originate from the ꢁisocopro-typeꢀ porphyrinogen intermediate. In order to
investigate the feasibility of alternative pathways for heme biosynthesis, the four type III pentacarboxylate isomeric porphyrinogens
were incubated with purified, cloned human coproꢀgen oxidase at 37 ꢀC with various substrate concentrations under initial velocity
conditions. Of the four isomers, only 5dab was a substrate for coproꢀgen oxidase and this gave dehydroisocoproporphyrin. The
structure of the related porphyrin tetramethyl ester was confirmed by proton NMR spectroscopy and mass spectrometry. The
m m
K value for protoꢀgen-IX formation from coproꢀgen, an indicator of molecular recognition, was similar to the K value for mono-
vinyl product formation with 5dab, although coproꢀgen-III has an approximately twofold higher Kcat value. Although 5dab is a
slightly poorer substrate than coproꢀgen-III, these results support the hypothesis that an abnormal route for heme biosynthesis is
possible in humans suffering from PCT or related syndromes such as hexachlorobenzene poisoning.
ꢁ
2005 Elsevier Ltd. All rights reserved.
3
,4
1. Introduction
chains to give methyl units (Scheme 1). At this stage,
is transferred into the mitochondria and undergoes
2
In mammals, the heme biosynthetic pathway takes place
via eight enzyme-mediated steps starting from glycine
and succinyl CoA, although in plants and bacteria the
first intermediate, d-aminolevulinic acid, is derived from
two oxidative decarboxylations by the enzyme coproꢀgen
oxidase to give protoꢀgen-IX (3). Further oxidation of
5
the hexahydroporphyrin 3 by protoꢀgen oxidase gives
protoporphyrin-IX, and subsequent iron(II) insertion
by ferrochelatase leads to heme b. Protoporphyrin-IX
is the precursor to many other heme type pigments,
and is also a key intermediate in the formation of the
1
,2
glutamate. The first macrocyclic intermediate in the
pathway, uroporphyrinogen-III (uroꢀgen-III; 1), is the
precursor to the vitamin B12 and related methylated
tetrapyrroles such as siroheme, as well as the hemes
1
,2
chlorophylls in photosynthetic organisms.
1
,2
and chlorophylls. The cytosolic enzyme uroꢀgen decar-
boxylase converts octacarboxylic acid 1 to the tetracarb-
oxylic acid coproporphyrinogen-III (coproꢀgen-III; 2) by
mediating the decarboxylation of four acetate side
In principle, uroꢀgen-III could be converted into coproꢀ-
gen-III by 24 different pathways involving up to 14 dif-
3
,4,6
ferent intermediates.
Depending upon whether the
initial acetate group undergoes decarboxylation on ring
A, B, C, or D, four heptacarboxylate intermediates
Keywords: Porphyrinogens; Isocoproporphyrins; Heme biosynthesis;
Coproporphyrinogen oxidase; Porphyria.
(heptaꢀs) may be formed (Chart 1). These, in turn, could
q
each be transformed at one of three different sites to
form a total of six different hexacarboxylate porphyrin-
ogens (hexaꢀs), and these could then be taken on via two
pathways each to give four pentacarboxylate species
(pentaꢀs). The four pentaꢀs only have one remaining
Normal and Abnormal Heme Biosynthesis. Part 5. For Part 4, see:
He, J.; Kaprak, T. A.; Jones, M. A.; Lash, T. D. J. Porphyrins
Phthalocyanines 2005, 9, in press.
*
0
Corresponding authors. Tel.: +1 309 438 8554; fax: +1 309 438
538; e-mail addresses: majone3@ilstu.edu; tdlash@ilstu.edu
5
968-0896/$ - see front matter ꢁ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.051

C. L. Cooper et al. / Bioorg. Med. Chem. 13 (2005) 6244–6251
–10
6245
8
3,11
CO H
thesis
and proton NMR spectroscopy.
d was identified as the major hepta component from a
In addition,
2
7
patient suffering from porphyria cutanea tarda (PCT)
CO H
2
1
1
and in incubations of porphobilinogen with chicken
red cell hemolysates containing a high concentration
HO C
N
N
CO H
2
2
HH
H H
1
0
Uro'gen
Decarboxylase
of sodium chloride. On the basis of these data, Jackson
postulated that uroꢀgen-III was transformed into coproꢀ-
gen-III by a selective route, where the D ring undergoes
the first decarboxylation followed by degradation at ring
A, then B, finally C (the ꢁclockwise decarboxylationꢀ
N
N
CO H
CO H
2
HO C
2
2
CH3
CO H CO H
2
2
CH3
3
1
(Uro'gen-III)
hypothesis). However, HPLC studies showed that mix-
N
H
HN
CO H
2
tures of heptaꢀs, hexaꢀs, and pentaꢀs were present in nor-
H H
12,13
N
N
mal urine,
and to add to the difficulty, were also
CH3
CH3
Copro'gen
Oxidase
formed in incubations of uroꢀgen-III with crude prepa-
CH3
6,14
rations of uroꢀgen decarboxylase.
However, at very
CH3
low substrate concentrations, uroꢀgen-III shows a higher
preference for 7d over the remaining heptaꢀs, and the
CO H CO2H
2
N
N
N
15
H
Proto'gen
Oxidase
2
(Copro'gen-III)
current consensus is that while a degree of random
processing may occur under some circumstances, the
clockwise decarboxylation pathway operates under
H
N
CH3
CH3
CH3
Fe2+
6,15
CH3
physiological conditions.
N
N
N
N
CO2H
CO2H
Ferro-
chelatase
Fe
Protoporphyrin-IX
Coproꢀgen-III is converted into protoꢀgen-IX (Scheme 2)
via the tricarboxylate intermediate harderoporphyrino-
gen (harderoꢀgen; 4), rather than the alternative species
CH3
CH3
1
6,17
isoharderoporphyrinogen (5).
There is no ambiguity
CO H CO2H
2
about whether the A or B ring is processed first for this
enzyme as 5 is a very poor substrate for coproꢀgen oxi-
dase and only porphyrins derived from 4 are observed
Chlorophylls
Heme b
1
8
Scheme 1. Biosynthesis of heme from uroporphyrinogen-III.
1
6,17
in enzyme incubation studies. Harderoporphyrin
was first isolated and characterized from the harderian
1
9
glands of rodents, but has been identified in feces
2
and other natural materials.
R3
Hepta's (4 type III isomers)
P
0
1
2
3
4
7
7
7
7
d R = Me; R = R = R = A
R2
2
1
3
4
P
a R = Me; R = R = R = A
A
B
In 1972, Elder reported the isolation of a series of tetra-
carboxylate porphyrins 6a–d (Chart 2) from porphyric
NH H N
H H
3
1
2
4
b R = Me; R = R = R = A
4
1
2
3
c R = Me; R = R = R = A
21
patients and rats poisoned with hexachlorobenzene.
The ethyl-substituted compound has the same molecular
N
N
R1
D
C
P
R4
Hexa's (6 type III isomers)
weight as coproporphyrin-III, and, for this reason, this
21,22
1
2
3
4
6
6
6
6
6
6
da R = R = Me; R = R = A
P
metabolite was named isocoproporphyrin.
structure of 6a has been rigorously demonstrated by
The
2
3
1
4
ab R = R = Me; R = R = A
Penta's (4 type III isomers)
2
4
1
3
ac R = R = Me; R = R = A
1
2
3
4
5
5
dab R = R = R = Me; R = A
degradation and proton NMR studies using europium
23
3
4
1
2
bc R = R = Me; R = R = A
2
3
4
1
24–26
abc R = R = R = Me; R = A
5acd R = R = R = Me; R = A
5bcd R = R = R = Me; R = A
shift reagents, as well as by total synthesis.
It
1
3
2
4
1
2
4
3
bd R = R = Me; R = R = A
was proposed that these unusual compounds are all de-
rived from the 3-vinyl species, dehydroisocoproporphy-
rin 6b, or the related porphyrinogen 7b, by bacterial
1
4
2
3
1
3
4
2
cd R = R = Me; R = R = A
Chart 1. The 14 possible intermediates between uroꢀgen-III and
coproꢀgen-III. The letter code designations refer to the rings (labeled
A–D in the porphyrin structure, but assigned in the structure codes
with the lower case letters a, b, c, and d) on which the acetate units
have been decarboxylated, while the numbers correspond to the
2
1,22
degradation in the gut.
Indeed, degradation of the
side chains for protoporphyrin-IX commonly gives rise
to related porphyrins such as deuteroporphyrin-IX,
2
0
pemptoporphyrin, mesoporphyrin-IX, etc. Evidence
for a fifth member of the series, the 3-acetylporphyrin
3
number of remaining carboxylate groups. Under physiological
conditions, the intermediates are 7d, 6da, and 5dab. Abbreviations
2
7
6e, has also been presented. The presence of an acetate
group on ring C suggests that 5dab has been premature-
ly metabolized by coproꢀgen oxidase. PCT and hexa-
chlorobenzene poisoning are both associated with an
accumulation of hepta-, hexa-, and pentacarboxylate
intermediates, and this may allow leakage of 5dab into
the mitochondria where coproꢀgen oxidase recognizes
2 2 2 2 2
for substituents: A = CH CO H; P = CH CH CO H.
acetate moiety and the final step for each would con-
verge to give coproꢀgen-III. Early studies demonstrated
that the process was sequential and the related porphy-
rin fractions corresponding to heptaꢀs, hexaꢀs, and pen-
2
8,29
this species and converts it into 7b.
It is possible that
7
taꢀs can be identified. In the 1970s, these porphyrin
7b could be further transformed into protoporphyrin-
IX, and this species represents a potential intermediate
in an abnormal ꢁisocoproꢀ pathway for heme biosynthe-
fractions were isolated from rats poisoned with hexa-
chlorobenzene and the corresponding methyl esters were
shown to be 7d, 6da, and 5dab, respectively, by total syn-
2
9
sis (Scheme 3). Pentacarboxylate porphyrinogen 5dab

6246
C. L. Cooper et al. / Bioorg. Med. Chem. 13 (2005) 6244–6251
CO H
2
CH3
CH3
CH3
CH3
CH3
CH3
NH HN
H H
NH HN
H H
CO H
CO H
2
2
O2
N
N
N
N
Copro'gen
Oxidase
CH3
CH3
CO2H
CO2H
CO2H
CO2H
2
(Copro'gen-III)
4
Hardero'gen
Copro'gen
Oxidase
O2
CO H
2
CH3
CH3
CH3
CH3
CH3
N
NH HN
H H
N
H H
H H
N
N
N
N
CH3
CH3
CH3
CO H
CO2H
CO2H
CO2H
2
5
(Isohardero'gen)
3 (Proto'gen-IX)
Scheme 2. Conversion of coproꢀgen-III to protoꢀgen-IX.
R
Me
R
Me
Me
P
Me
P
Me
Me
Me
Me
Me
Me
P
Me
P
Copro'gen
P
N
H
N
N
HN
H
H
N
N
N
N
NH HN
H H
Oxidase
H
H H
O2
NH HN
N
N
H
N
N
A
(mitochondria)
A
Me
A
A
P
P
P
P
7
b
P
P
P
P
5
dab
Dehydroisocopro'gen
6
7
a. R = Et; b. R = CH=CH ; c. R = H
2
Uro'gen
Decarboxylase
(cytoplasm)
d. R = CH(OH)CH ; e. R = COCH
3
Chart 2. Structures of the isocoproporphyrin series and the related
porphyrinogens. It should be noted that porphyrins 6 were synthesized
and characterized as the corresponding tetramethyl esters. Abbrevia-
Me
Me
P
Me
P
Me
Copro'gen
Oxidase
N
N
N
H H
H H
NH HN
H H
2 2 2 2 2
tions: A = CH CO H; P = CH CH CO H.
N
O2
N
N
Me
Me (mitochondria)
Me
Me
has been shown to be converted to 7b by rat liver prep-
arations of coproꢀgen oxidase, but further conversion to
P
P
P
P
2
8
3 (Proto'gen-IX)
4 (Hardero'gen)
a divinyl product is not observed. Synthetic iso-
coproporphyrinogen (7a) is also not a substrate for the
rat liver enzyme. However, 7a, 7b, and 7c have been
shown to be metabolized by the uroꢀgen decarboxylase
present in chicken red cell hemolysates to give hard-
eroꢀgen or related tricarboxylate porphyrinogens, and
these can then be further processed by coproꢀgen oxi-
Scheme 3. Proposed abnormal pathway for heme biosynthesis.
decarboxylase activity suggests that this pathway could
3
operate in yeast as well.
0
2
9
dase. These data show that an abnormal pathway is
possible for protoꢀgen-IX production following the
sequence 5dab to 7b to harderoꢀgen to protoꢀgen-IX
The observations noted above, together with extensive
studies using substrate analogues, has allowed the devel-
opment of a model for substrate binding in coproꢀgen
oxidase (Fig. 1). A sequence of substituents R Me–P
Me–P, where R is a small nonpolar group such as H,
Me, Et, or vinyl, and P = propionate, appears to be
needed for substrate binding and oxidative decarboxyl-
(
Scheme 3), although the intermediates would have to
be shuttled back and forth between the mitochondria
and cytoplasm at each step. The formation of dehy-
droisocoproporphyrinogen by mutants of Saccharomy-
ces cerevisiae that are partially deficient in uroꢀgen
3
1,32
ation of the first propionate residue.
Changing

C. L. Cooper et al. / Bioorg. Med. Chem. 13 (2005) 6244–6251
6247
rin methyl esters using normal phase HPLC. Repeated
experiments demonstrated that only 5dab was a sub-
strate for coproꢀgen oxidase, and the three isomeric type
III porphyrinogens showed no indication of product
formation. These data indicate that acetate moieties
cannot be tolerated in place of group R at site Z in
the active site model, or in place of the first methyl unit
in the sequence. On the other hand, 5dab proved to be a
very good substrate giving dehydroisocoproporphyrino-
gen 7b as the only product, and in agreement with earlier
investigations no further metabolism of the B ring
propionate residue was observed.
Y
X
CO H
CH3
2
CO2H
N
H
N
CH3
H
H N
CH3
H
N
CO2H
Z
CH3
In order to further assess how good a substrate 5dab is
for cloned human coproꢀgen oxidase, more detailed
kinetic studies were conducted. Figure 2a shows the
CO H
2
Figure 1. Proposed model for the active site of coproꢀgen oxidase.
apparent initial velocity (v ) for the accumulation of to-
0
tal, divinyl (3), and monovinyl (4) products after incuba-
tion of 7.5 lg of the purified enzyme with the authentic
substrate coproꢀgen-III (1 lM). Figure 2b shows the
monovinyl product 7b formed from 5dab (1 lM). Total
product was calculated as the sum of the monovinyl and
divinyl products using coproꢀgen-III. These data are
from three replicate incubations, and the range of high
and low values about the mean was 5%. The equations
for the apparent linear portion of the lines (from 0 to
2 min for coproꢀgen-III and 5 min for 5dab) were evalu-
ated by linear regression analysis. The initial velocity for
coproꢀgen-III was more than twofold faster than that of
5dab (19.8/min and 8.2/min, respectively). Figure 3a
either of the propionate side chains to butyrate,
CH(CH )CH CO H, or CH CH(CH )CO H leads, at
3
2
2
2
3
2
3
3
best, to much reduced activity. The proposed model
(
Fig. 1) identifies three significant regions within the ac-
5
,32,34–36
tive site for coproꢀgen oxidase.
Site X recognizes
the second propionate group, probably via a salt bridge
interaction, Y represents the catalytic site, while region
Z can only tolerate small nonpolar groupings. Although
this model has been very successful in predicting the sub-
3
4–36
strate specificity for coproꢀgen oxidase,
it is not clear
what types of minor changes can be tolerated in the
peripheral sequence. R cannot be a propionate group,
but a CH = CH–CH3 unit does appear to fit while
shows v (nmol product/min/pmol enzyme) as a function
0
of substrate concentration from 0.01 to 6.1 lM using
3
3
C(CH ) = CH is poorly tolerated. The observation
3
coproꢀgen-III as substrate. Figure 3b shows v as a func-
2
0
that 7a or 7b are not substrates for rat liver coproꢀgen
oxidase shows that the second methyl group in the se-
quence cannot be replaced by an acetate unit, but it is
not clear whether acetate groups could be present in
place of the first methyl group or instead of the usual
nonpolar groupings for R. As four pentaꢀs can be
formed from uroꢀgen-III, alternative ꢁneocoproꢀ path-
ways for heme biosynthesis could exist, and to our
knowledge this possibility has never been tested. In addi-
tion, studies of this type using purified human coproꢀgen
oxidase have not been conducted previously. In this
paper, we report the first studies of the abnormal ꢁisoco-
proꢀ pathway using cloned human coproꢀgen oxidase
and investigate the metabolism of all four type III pen-
tacarboxylate porphyrinogens.
tion of 5dab substrate concentration from 0.01 to
6.6 lM. The kinetic constants (K , Kcat, and Kcat/K ),
m
m
using both substrates, are shown in Table 1. When com-
paring total product, coproꢀgen-III has about a twofold
lower Km than 5dab, approximately a twofold higher
Kcat, and about a fourfold higher Kcat/Km ratio. Even
though the values for the same kinetic constants are
3
,4
within an order of magnitude of each other, the Kcat
K values, a measure of catalytic efficiency, indicate that
/
m
the sequential oxidative decarboxylations of the C-III
substrate are fourfold more efficient than the single oxi-
dative decarboxylation of 5dab. Hence, even though the
presence of an acetate group on ring C does not signif-
icantly inhibit metabolism, it still appears to have a
small detrimental influence.
In order to rigorously confirm that dehydroiso-
coproporphyrin 6b had in fact been generated from
2
. Results and discussion
5dab, a larger scale experiment was carried out and
Highly purified enzyme (12 mg/L of cell culture, single
band by SDS–PAGE, MW of 37,000) was obtained
for these studies (data not shown). Pentacarboxylate
porphyrinogens 5dab, 5abc, 5abd, and 5acd were pre-
pared by hydrolysis of the corresponding porphyrin
methyl esters and reduction with 3% sodium amalgam
the isolated porphyrin product was characterized by
proton NMR spectroscopy and mass spectrometry.
The proton NMR spectrum of the isolated product
(Fig. 4a) shows the presence of seven methyl groups be-
tween 3.6 and 3.8 ppm corresponding to the four methyl
esters and the three porphyrin methyl groups. The vinyl
moiety was evident from the three doublet of doublets at
6.17, 6.36, and 8.28 ppm, while the three propionate side
chains afforded two 6H multiplets at 3.3 and 4.5 ppm,
and the methylene component of the acetate group gave
a 2H singlet at 5.1 ppm. Finally, the internal NHs were
5
under standard conditions. The pentacarboxylate por-
phyrins were previously synthesized via a,c-biladiene
4
intermediates. Following incubation of the porphyrino-
gens with the cloned human enzyme preparations, the
metabolites were analyzed as the corresponding porphy-

6248
C. L. Cooper et al. / Bioorg. Med. Chem. 13 (2005) 6244–6251
a
80
70
6
0
0
5
40
30
20
10
0
0
5
10
15
20
25
Time (min)
b
80
70
6
0
0
5
40
30
20
10
0
0
5
10
15
20
25
30
35
Time (min)
Figure 2. Time course experiments for coproꢀgen-III (a) and 5dab (b) showing the the percent product formation versus time. For (a), triangles
represent total % product accumulation, squares represent % divinyl product accumulation, and circles represent % monovinyl product formation
from coproꢀgen-III. For (b), circles indicate the % monovinyl product formed from 5dab. All values are the mean of three replicate incubations. The
range of high and low values about the mean was 5%.
evident as a 2H singlet at À3.7 ppm, while the meso-
3. Conclusion
protons gave three singlets in the ratio of 2:1:1 near
1
0.2 ppm. In the original structure determination of iso-
The metabolism of 5dab by purified human coproꢀgen
oxidase has been demonstrated, but the other three type
III pentacarboxylate porphyrinogens are not metabo-
lized by this enzyme. Porphyrinogen 5dab appears to
bind to the active site with good affinity, having a Km
of 1.3 lM, which is comparable to the Km of coproꢀ-
gen-III, which is 0.54 lM, and the kinetic values show
that this pentacarboxylate substrate is a good substrate
when considering the turnover rate (Kcat). Hence,
the feasibility of an abnormal ꢁisocoproꢀ pathway for
heme biosynthesis is supported by these data, while
the possibility of other ꢁneocoproꢀ pathways can now
be dismissed.
coproporphyrin, europium shift reagents were used to
show how the ester groups were arranged around the
periphery of the macrocycle. When two ester groups
flank a given meso-proton, the shift reagent associates
relatively strongly at that site causing a significant
downfield shift to the nearby meso-resonances. Addition
of Eu(fod) to our NMR solution in CDCl showed that
two of the meso-protons were shifted downfield and sig-
nificantly broadened, while the other two resonances
were essentially unaffected (Fig. 4b). This is exactly the
result expected as two of the meso-protons are surround-
ed by two ester units in 6b, while the other two have
no ester moieties nearby. FAB MS provided further
3
3
confirmation of the structure and gave the expected
[
In disease states where 5dab is released prematurely
from uroꢀgen oxidase, it can compete with the normal
+
M+H] ion at m/z 709.

C. L. Cooper et al. / Bioorg. Med. Chem. 13 (2005) 6244–6251
6249
a
0.0012
0.001
0.0008
0.0006
0.0004
0.0002
0
0
1
2
3
4
5
6
7
[S]µM
b
0.0007
0
0
0
0
0
0
.0006
.0005
.0004
.0003
.0002
.0001
0
0
1
2
3
4
5
6
7
[S]µM
Figure 3. (a) Initial velocity curve as a function of substrate concentration with coproꢀgen-III. (b) Substrate concentration curve with 5dab. All values
are the mean of three replicate incubations. The range of high and low values about the mean was 5%. See Figure 2 for key.
Table 1. Kinetic constants of coproꢀgen oxidase for monovinyl,
divinyl, and total product accumulated
IX and ultimately to heme b. This convoluted process
greatly increases the likelihood that spontaneous oxida-
tion to the porphyrin form will occur. As the aromatic
forms are not recognized by either of the enzymes, these
abnormal metabolites would accumulate, exacerbating
symptoms such as light sensitivity for porphyric
patients.
Substrate
K
m
K
cat
Kcat/K
m
À1
(
lM)
(min )
Monovinyl product
C-III
dab
0.97
1.3
0.20
0.54
0.21
0.42
5
Divinyl product
Total product
C-III
0.55
0.75
1.4
C-III
5
0.54
1.3
0.97
0.54
1.8
0.42
dab
4. Experimental
4.1. Isolation and purification of coproꢀgen oxidase
substrate 2 for the active site of coproꢀgen oxidase and
generate dehydroisocoproporphyrinogen. However, fur-
ther metabolism to protoꢀgen-IX can only occur if the
porphyrinogen migrates back into the cytoplasm to be
acted upon by uroꢀgen decarboxylase to form harder-
oꢀgen, followed by transport back into the mitochondria
so that the porphyrinogen can be taken on to protoꢀgen-
Escherichia coli, containing the gene for the human
enzyme, were grown overnight at 37 ꢀC with shaking
at 250 rpm, in 1-L cultures using a medium consisting
of 10 g tryptone (Fischer Chemical Company), 5 g yeast
extract (Sigma Chemical Company), and 10 g NaCl
per liter nanopure water. Cells were isolated by

6250
C. L. Cooper et al. / Bioorg. Med. Chem. 13 (2005) 6244–6251
a
8.5
8.0
6.5
ppm
10.5
10.0
10
9
8
6
5
4
3
–3
ppm
b
11.5
11.0
10.5
10.0
8.5
ppm
δ
3
Figure 4. (a) 400 MHz proton NMR spectrum in CDCl of dehydroisocoproporphyrin tetramethyl ester isolated from incubations of 5dab with
3
coproꢀgen oxidase. (b) Downfield region for the same sample in the presence of the europium shift reagent Eu(fod) . Two of the four meso-protons
were shifted downfield producing broadened resonances while the other two were essentially unaffected.
centrifugation and lysed using the French hydraulic
pressure cell (Sim-Aminco Spectronic Instruments).
The enzyme, which has the 6 · histidine tag, was
acetate followed by extraction and methyl esterification
overnight. In all experiments, a zero incubation control
(addition of acetic acid/ethyl acetate before addition of
substrate) was also performed for comparison. Follow-
ing neutralization and extraction of the porphyrin meth-
yl esters, they were analyzed using high pressure liquid
chromatography (HPLC; Beckman System Gold). A
normal phase column (Beckman Silica 5 l,
4.6 mm · 25 cm) was used with a solvent of 35/65 (v/v)
ethyl acetate/cyclohexane for C-III and 50/50 (v/v) for
the pentacarboxylates at a flow rate of 1.3 mL/min;
elutes were evaluated spectrophotometrically at a wave-
length of 404 nm. Data were analyzed using the Gold
Nouveau Software and reported as percent product or
subsequently converted to the units of Medlock and
2
+
isolated by the Ni affinity procedure of Medlock and
3
7
Dailey. Protein was analyzed by sodium dodecyl sul-
fate polyacrylamide gel electrophoresis (SDS-PAGE)
3
8
using the Laemmli method to test the purity and
apparent molecular weight of the enzyme. The Bradford
3
9
Protein Assay was used to evaluate the concentration
of the enzyme and bovine serum albumin (BSA) was
used as the standard.
4
.2. Enzyme assay
4
0
Using the micromethod of Jones et al., coproꢀgen-III,
dab, 5abc, 5bcd, or 5cda were incubated with the co-
proꢀgen oxidase (300 lL of 25 lg/mL) at 0, 0.25, 0.5,
.75, 1, 2, 3, 5, 10, 20, or 30 min for one set of experi-
3
7
5
Dailey. Incubations with the other three pentacarb-
oxylate porphyrinogens showed no detectable product
under any conditions tested.
0
ments using 1 lM substrate. In another set of experi-
ments, substrate concentration was varied from 0.01 to
4.3. Isolation of dehydroisocoproporphyrin tetramethyl
ester
6
1
.1 lM, while holding the incubation time constant at
min for coproꢀgen-III, and from 0.01 to 6.6 lM for
an incubation time of 3 min for 5dab. The reactions
In order to collect sufficient product for proton NMR
characterization, a 1 L scale incubation was performed.
were stopped with addition of 3/7 (v/v) acetic acid/ethyl

C. L. Cooper et al. / Bioorg. Med. Chem. 13 (2005) 6244–6251
6251
Cloned coproꢀgen oxidase (25 mg) was isolated from 2 L
of E. coli cells and diluted in 250 mM imidazole buffer
10. Battersby, A. R.; Hunt, E.; McDonald, E.; Paine, J. B.,
III; Saunders, J. J. Chem. Soc., Perkin Trans. 1 1976,
1
008.
(
pH 7.0) to give 1 L. This preparation was incubated
1
1
1. Jackson, A. H. Sem. Hematol. 1977, 14, 193.
2. Smith, S. G.; Rao, K. R. N.; Jackson, A. H. Int.
J. Biochem. 1980, 12, 1081.
with 1 mg of porphyrinogen 5dab for 1 h at 37 ꢀC with
gentle shaking. The incubation was terminated by the
addition of 2.85 L 3/7 (v/v) acetic acid/ethyl acetate
and extracted as described previously. Following esteri-
fication with 5% sulfuric acid–methanol at room temper-
ature for 16 h, HPLC showed that the extract contained
1
3. Lim, C. K.; Rideout, J. M. J. Liq. Chromatogr. 1983, 6,
1
969; Lim, C. K.; Rideout, J. M.; Wright, D. J.
J. Chromatogr. 1983, 282, 629; Li, F.; Lim, C. K.; Peters,
T. J. Biochem. J. 1987, 243, 621; Lim, C. K.; Li, F.; Peters,
T. J. Biochem. J. 1987, 247, 229.
8
silica eluting with 3/7 ethyl acetate/toluene gave the
0% product. Purification by flash chromatography on
14. (a) Luo, J.; Lim, C. K. Biochem. J. 1990, 268, 513; (b) Luo,
J.; Lim, C. K. J. Chromatogr. 1991, 566, 409.
1
3
-vinylporphyrin. H NMR (400 MHz, CDCl ): d
3
1
1
5. Luo, J.; Lim, C. K. Biochem. J. 1993, 289, 529.
6. Elder, G. H.; Evans, J. O.; Jackson, J. R.; Jackson, A. H.
Biochem. J. 1978, 169, 215.
À3.67 (2H, s), 3.29–3.38 (6H, m), 3.65 (3H, s), 3.67
(
(
3H, s), 3.69 (3H, s), 3.70 (3H, s), 3.71 (6H, s), 3.77
3H, s), 4.42–4.50 (6H, m), 5.12 (2H, s), 6.17 (1H, dd,
1
7. Jackson, A. H.; Jones, D. M.; Philip, G.; Lash, T. D.;
Battle, A. M. del C.; Smith, S. G. Int. J. Biochem. 1980, 12,
J = 1.5, 11 Hz), 6.36 (1H, dd, J = 1.5, 17 Hz), 8.28
1H, dd, J = 11, 17 Hz), 10.17 (2H, s), 10.18 (1H, s),
0.23 (1H, s). HRMS (FAB): Calcd for
C H N O + H: 709.3237. Found: 709.3236.
(
1
6
81.
1
8. (a) Cavaleiro, J. A. S.; Kenner, G. W.; Smith, K. M. J.
Chem. Soc., Perkin Trans. 1 1974, 1188; (b) Evans, N.;
Jackson, A. H.; Matlin, S. A.; Towill, R. J. Chromatogr.
4
0
44
4
8
1
976, 125, 345.
Acknowledgments
19. Kennedy, G. Y.; Jackson, A. H.; Kenner, G. W.; Suckling,
C. J. FEBS Lett. 1970, 6, 9.
This work was supported by the National Institutes of
Health under Grant No. 1 R15 GM/OD52687-01A1
and the National Science Foundation under Grant
No. CHE-0134472. The cDNA encoding 6 · -histidine-
tagged human coproꢀgen oxidase was a generous gift
from Dr. H. A. Dailey of the University of Georgia
20. Jackson, A. H.; Rao, K. R. N.; Smith, S. G.; Lash, T. D.
Biochem. J. 1985, 227, 327.
2
1. (a) Elder, G. H. Biochem. J. 1972, 126, 877; (b) Elder, G.
H. J. Clin. Pathol. 1975, 28, 601.
2. Elder, G. H. Sem. Hematol. 1977, 14, 227.
2
2
3. Stoll, M. S.; Elder, G. H.; Games, D. E.; OꢀHanlon, P.;
Millington, D. S.; Jackson, A. H. Biochem. J. 1973, 131,
(
Athens, GA).
4
29.
2
4. Baptista de Almeida, J. A. P.; Kenner, G. W.; Rimmer, R.;
Smith, K. M. Tetrahedron 1976, 32, 1793.
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