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OCR for page 66
PART II. BIOSYNTHESIS OF HEMOGLOBIN
THE BIOSYNTHESIS OF PORPHYRINS
DAVID SHEMIN
The over-all pathway of porphyrin synthesis in the cell is now known. This
paper will first summarize this pathway, with the pertinent evidence, and
then consider some further data which may eventually elucidate the details
of those reactions which are concerned with porphyrin synthesis.
The elucidation of the pathway of porphyrin synthesis was greatly aided,
after the initial observations,~~~~3 by the early finding of an in vitro system
capable of synthesizing this complicated-looking molecule from its compara-
tively simple precursors. It was found that both avian erythrocytes4 and
mammalian reticulocytes~ can effect this synthesis in vitro. As these systems
were investigated it was found later that hemolyzed preparatiorls6~ ~ and
extracts of avian erythrocytesS under proper conditions could also synthesize
the porphyrin molecule.
Only two precursors, glyc~ne and succinate, are required for all the atoms
.
of the porphyrin molecule. this was demonstrated by incubating duck ery-
throcytes with labeled substrates and then degrading the porphyrin molecule
ill a manner by which each carbon atom from a specific position could be
isolated. It was found that the carbon atoms of the substrates occupy particu-
lar positions in the porphyrin molecule.9~~0
Heme synthesized from glycine-2-C24 was shown to contain eight radio-
active 4~~~ carbon atoms in specific positions.9 i~ Samples of heme, synthesized
from Calf methyl and C7^J' carboxyl-labeled acetate, which were degraded, re-
vealed a labeling pattern from which it was concluded that the acetate was
converted to a four-carbon atom unsymmetrical compound via the citric acid
cycled Further, it was concluded that this "active" succinate condensed with
glycine, in some unknown manner, to form a precursor pyrrole. The relation-
ship of porphyrin formation to the citric acid cycle is shown in figure 1.
This relationship was documented by studies in which succinate- 1,4-C',
succinate-2,3-C2'', a-ketoglutarate-l,2-Cl;, a-ketoglutarate-5-C7'i arid pri-
mary carboxyl-labeled citrate were the substrates.6 13 In each of the experi-
ments the predicted carbon atoms in the porphyrin contained the Call.
The condensation of glycine and the active succinate (Reaction D, fin. 1)
, . . . ~ . , .
~ , O
divas then Investigated. An consideration of the possible methods of condensa-
tion of succinate and glycine which would give rise to a product from which
a pyrrole could reasonably be synthesized, a mechanism for detaching the
~ This work was supported by grants from the National Institutes of Health, IJnited
States Public Health Service (A-1101, C-8), from the American Cancer Society, from
the Rockefeller Foundation and from the Williams-Waterman Fund.
66
OCR for page 67
BIOSYNTHESIS OF PORPHYRINS SHEMIN
< . .
| (F)
~7
TRICARBOXYLIC ACID CYCLE
(F)
(A)
> oc-Ketoglutarate >
Succinyl derivative
(D ) 1 + Glycine
(E)
Pyrroles ~ Protoporphyrin
(F)
Succinate
1
FIG. 1. The relationship of the tricarboxylic acid cycle and porphyrin formation.
carboxyl group of glycine from its a-carbon atom must also be taken into
account. This must be considered since the carboxyl group of glycine is not
utilized for porphyrin formation and in the initial condensation of glycine with
succinate, the whole molecule of glycine is involved. The condensation of succi-
{T ~ I C A R BOX Y L I C\
ACID
GYCLE
~SU CC I NY L)
UREIDO group of purines, SlJCGlN4~E I
formate, etc. ~ `~'
c(- 1< ETO G LU T A R A T E
~ f G/ycine
-\ /SUCCI NATE- H
th e 1-ca rbon ato m toy G L YC I N E HOOC-CH2- CH2-C - C - COOH
~ I _
gOOC-CH2-CH2-C- CHO
o
k e t o - 9 ~ u t o r a I d e h y d e
/ ~ A ~ ~ ~
1501
HOOC- CH2_CH2_C_C0OH
o
Ct - keto-glutaric acid
~-amino-~-keto ad ~ pic acid ( I
J-co2
HOOC-CH2-CH2- ~C, -CH2NH2
o
c; - amino - levulinic aci d ( I: )
P O R P H Y R I I\J
FIG. 2. The Succinate-G!ycine cycle: a pathway for the metabolism of glycine.
OCR for page 68
68 PART II. BIOS YNTHESIS OF HEMOGLOBIN
nate on the a-carbon atom of glycine to form a-amino-~-keto adipic acid would
appear to be in agreement with the experimental Endings and conclusion (fig.
29. The compound formed, a p-keto acid, could then undergo decarboxylation
readily and thus provide a mechanism by which the carboxyl group is detached
from its a-carbon atom. :Further, the product of decarboxylation would be an
amino ketone, b-2minolevulinic acid. Condensation of two moles of this latter
compound by a Kr~orr type of condensation would give a reasonable mechanism
for the formation of a pyrrole in which the carbon atoms of glycine and
succinate would be in the previously found positions (fig. 3~.
In order to test this postulate, hemolyzates of duck erythrocytes were in-
c~bated with b-aminolevulinic acid-5-C 5 and with b-aminolevulinic acid-
1,4-C~. i~ Not only were the heme samples much more radioactive than com-
parable samples synthesized from radioactive glycine and succinate, but the
labeling pattern in the heme was the same for both b-aminolevulinic acid-
5 C25 and glycine-2-C~-;, and for both b-aminolevulinic acid-1,4-C' and
succinic acid-1,4-C-5. i;~~~0 These experiments demonstrated that 6-amino-
levulinic acid is an intermediate in porphyrin synthesis. This conclusion was
supported by the experiments of Neuberger and Scotti' and by Dresel and
Falk.~S Furthermore, it was subsequently demonstrated that fractions ob-
tained from liveri9~~° and avian red blood cells"'' catalyze the formation of
the mono-pyrrole, porphobilinogen':3~''4 (fig. 3), which divas previously shown
try be an intermediate in the formation of porphyrin."
The above is a summary of the synthesis of porphyrin from its precursors,
glycine and succinate. We may now consider some experiments which were
carried out in order to shed some light on the intimate details of some of
the steps.
The Formation of b-Aminolev~linic Acid. The synthesis of b-aminole-
vulinic acid from glycine and succinate appears to be a rather complicated
reaction in regard to the nature of the activated derivatives and to the bio-
logical system. Whereas hemolyzates of avian erythrocytes can synthesize
COOH
H2
l H2
COO H
COGH CH2
1
COOH
~ H2
c-2 + ;~° -2~0 ~ ~ PROTO-
-C 0 ~ H2
H2N
5-A MING
LEVULINIC aCID
(II) + (mu)
~ N
NH2 H
P R E C U RSOR
P Y PRO LE
FIG. 3.—A mechanism for the formation of the monopyrrole, porphobilinigen, by
condensation of two moles of b-aminolevulinic acid. The carbon atoms bearing the
closed circles were originally the of-carbon atom of glycine.
OCR for page 69
BIOSYNTHESIS OF PORPHYRINS—SHEMIN
69
porphyrins from al; cine and succinate, preparations obtained by homogeni-
zation, freezing and thawing, acetone powders, and extracts can only utilize
b-aminolevulinic acid as a substrate for protoporphyrin synthesists i;' Ap-
parently, the system responsible for the synthesis of 6-aminolevulinic acid is
quite labile and complex.
We found several years ago that certain compounds would inhibit the
formation of b-aminolevulinic acid. It was found that cysteine, pyruvate, and
acetate-'0 would inhibit b-aminolevulinic acid formation. This was ascertained
from experiments which demonstrated that porphyrin synthesis was inhibited
by addition of these compounds when glycine and succinate were the sub-
strates and not when b-aminolevulinic acid Bras the substrate. We have found
recently that not only can pyridoxal phosphate increase the synthesis of
porphyrins as demonstrated by Schulman and Richert,-` but that the cysteine
inhibition can be overcome by this coenzyme.'S These experiments point to a
necessary activation of the glycine. We have more recently found that forma-
tion of b-aminolevulinic acid can be markedly inhibited by aza-L-serine.'0
This latter inhibition was not overcome by the addition of glutamine.~° It
may be worth noting that azaserine has no inhibitory effect on the conversion
of b-aminolevulinic acid to heme. Surprisingly, the addition of 6-diazo-5-
oxo-L-norleucine, which is a more effective inhibitor than azaserine in purine
ring synthesis,:~° has no inhibitory influence on the formation of b-amino-
levulinic acid. At this moment it is difficult to describe definitely the details
concerned with glycine activation, especially in consideration of the above
experiments. The activation of succinate is as yet to be elucidated. The ex-
periments which were done with labeled acetate definitely established the
formation of an unsymmetrical succinate and it was suggested, at that time,
that this may be a succinyl coenzyme derivative.~° As yet the nature of this
derivative has not been established.
We haste carried out model organic experiments in which glycine and
succinate were activated in order to see if b-aminolevulinic acid can be formed
under relatively mild condition. Glycine was converted into an oxazolone
derivative and succinate was in the form of its anhydride. Base-catalyzed
condensation of these molecules occurred and b-aminolevulinic acid was
demonstrated after hydrolysis of the condensed product.3i
The Formation of Porphobilinogen from b-Aminole~linic Acid. The
enzymatic formation of porphobilinogen from two moles of b-aminolevulinic
acid requires that two different types of reactions should occur; an aldol type
condensation and a Schiff base type linkage. Gibson, Neuberger and Scott'°
have obtained no evidence that these enzyme preparations consisted of two
enzymes. The enzyme concerned with porphobilinogen may, however, only
catalyze one of these reactions, e.g., the aldol condensation. This reaction
only may need the catalysis, for once this occurs the Schiff base reaction
may occur spontaneously. In order to shed some light on the mechanism of
OCR for page 70
70
PART II. BIOSYNTHESIS OF HEMOGLOBIN
porphobilinogen formation we have investigated model organic reactions.
Scott3> and wee have found that b-aminolevulinic acid in alkali under anaerobic
conditions would to a small extent be converted to porphobilinogen. In order
to increase the yield and if possible to isolate intermediates which could sub-
sequently be converted to porphobilinogen and to attempt to understand the
formation of porphobilinogen, we have acylated the amino group of b-amino-
levulinic acid and then subjected these derivatives to alkaline and anaerobic
~ . .
conditions.
An acid Group on the amino croup which is not readily hvdrolvzed bv
~ =~ ~ — ~ > ~ 1~ — ~ — —~ — — —~ — — — ~
~ . .. .., . . . . ~ . ~ . . . .
alkali at room temperature will hinder the formation of pyraz~ne derivatives,
while permitting an aldol condensation to occur between two molecules. Sub-
sequent hydrolysis of the ac`,;1 Groups would permit a Schiff base reaction and
~ J J J D ~ - ~
the product WOU1d be a pyrrole. 1 he structure of the pyrrole will depend on
the initial carbon atoms involved in the aldol condensation. N-Acetyl b-amino-
levulinic acid subjected to the conditions mentioned above yielded, after
several days, products which on exposure to air were converted to an intense
red pigment and which gave an intense color with Ehrlich's reagent. The
color intensity obtained with Ehrlich's reagent indicated a very high yield of
these compounds. The structure or structures of the product await elucidation.
N-Phthalimido derivatives which were subjected to the same conditions may
yield open chain condensation products because of the resistance of the
phthalimido grouping to complete alkaline hydrolysis.3i
The Formation of Porphyrins frown the mono-pyrrole, porphobilinogen.
The mechanism of the conversion of the mono-pyrrole, porphobilinogen, to
the biological functioning porphyrin (III isomer) has not been elucidated.
We have suggested a mechanism which is based on the organic experiment of
Corwin and collaborators.33~34 Condensation of three moles of the porpho-
bilinogen could lead to a tripyrrylmethane compound as represented in figure
4. The tripyrrylmethane then breaks down to a dipyrrylmethane and a mono-
pyrrol. The structure of the dipyrrylmethane is dependent on the place of
Ac P Ac P ~ Ac P Be P
~ A ~ f, .~ . -2~H2
CH2—NH2 Ac P P Ac
FIG. 4.—A mechanism of porphyrin formation from the monopyrrole. Ac—Acetic
acid side chain; P Propionic acid side chain; ~ a-carbon atom of glycine and
6-carbon atom of 6-aminolevulinic acid.
OCR for page 71
BIOSYNTHESIS OF POF(PlIVRINS—SHEMIN
71
splitting. Art A split would give rise to dipyrrylmethane A, and a B split
should give rise to dipyrrylmethane B. Cor~densation of a mole of A and a
mole of B would give rise to a porphyrin of the III series. In the formation
of the porphyrins of the III series it can be seen from figure 4 that it is
necessary to lose a one-carbon atom compound since there are three amino-
methyl side chains and only two are required to condense the two dipyrroles
to the porphyrin structure. Consistent with this hypothesis is our finding that
on the conversion of porphobilinogen to porphyrins, either by heating under
acid conditions or by enzymatic conversion in cell-free extracts, formaldehyde
i, formed.~5
The formation of protoporphyrin and heme can occur in a cell-free extract
of duck erythrocytes. After incubation of cell-free extracts, obtained by
centrifugation at 100,000 g, with 6-aminolevulinic acid-5-C ;, the isolated
bemire was radioactive. The radioactivity was constant after several re-
crystallizations. The hemin was then subjected to a chemical degradation in
order to isolate methylethvlmaleimide and hematir~ic acid. The methyl-
ethylmaleimide can only arise from pyrrole rings A and B of protoporphyrir~.
It was found that the sample of methylethylmaleimide was radioactive and
equal to that of the hematinic acid. Furthermore, the sum of the radioactivity
of the methylethylmaleimide and hematinic acid was equal to the value cal-
culated from the radioactivity of the hemin.35
The picture or porphyrin synthesis which has been summarized emphasizes
the general concepts which have emerged from the biochemical studies carried
out during the past two decades: the relative simplicity of the reactions; the
relative simplicity arid availability of the substrates utilized for the synthesis
of complicated structures; and the biochemical unity in living matter. Pro-
toporphyrin is synthesized from two simple and readily available compounds,
glycine and succir~ate, by rather simple reactions and the synthesis is very
closely linked to the main energy-yieldir~g reactions of most cells. Further, it
appears that all porphyrins in nature, including chlorophyll, in all different
types of cells are synthesized by the same basic pathway. The different por-
phyrins merely arise by modifications occurring in the side chains ire the Q-
positions of the pyrrole units. In further support of this conclusion it is worth
mentioning our recent studies ore the biosynthesis of vitamin Bee. The struc-
ture of the vitamin has recently been formulated to contain a porphyrin-like
component.36~3' We have found that b-aminolevulinic acid is readily utilized
for the synthesis of vitamin Bed :3s and that the predicted carbon atoms of
the vitamin synthesized from b-aminolevulinic acid-l,4-C7; contained the
radioactivities.~0
REFEFtEN CES
1. Shemin, D., and Rittenberg, D.: The utilization of glycine for the synthesis of a
porphyrin, J. Biol. Chem., 159: 567, 1945.
OCR for page 72
72 PART II. BIOSYNTHESIS OF HEMOGLOBIN
2.
7.
a.
Shemin, D., and Rittenberg, D.: The biological utilization of glycine for the syn-
thesis of the protoporphyrin of hemoglobin. J. Biol. Chem., 166: 621, 1946.
Shemin, D., and Rittenberg, D.: The life span of the human red blood cell, J.
Biol. Chem., 166: 627, 1946.
4. Shemin, D., London, I. M., and Rittenberg, D.: The synthesis of protoporphyrin
in vitro by red blood cells of the duck, J. Biol. Chem., 173: 799, 1948; 183: 757,
1950.
SO London, I. M., Shemin, D., and Rittenberg, D.: Synthesis of heme in Vitro by the
immature non-nucleated mammalian erythrocyte, .~. Biol. Chem., 173: 797, 1948;
183: 749, 1950.
6. Shemin, D., and Kumin, S.: The mechanism of porphyrin synthesis. The formation
of a succinyl intermediate from succinate, J. Biol. Chem., 198: 827, 1952.
London, I. M., and Yamasaki, M.: Heme synthesis in non-intact mammalian and
avian erythrocytes, Federation Proc., 11: 250, 1952.
Shemin, D., Abramsky, T., and Russell, C. S.: The synthesis of protoporphyrin
from 6-aminolevulinic acid in a cell-free extract, J. Am. Chem. Soc., 76: 1204,
1954.
9 Wittenberg, J., and Shemin, D.: The location in protoporphyrin of the carbon
atoms derived from the or-carbon atom of glycine, J. Biol. Chem., 185: 745, 1950.
10. Shemin, D., and ~rittenberg, J.: The mechanism of porphyrin formation. The role
of the tricarboxylic acid cycle, J. Biol. Chem., 192: 315, 1951.
11. Radin, N., Rittenberg, D., and Shemin, D.: The role of glycine in the biosynthesis
of heme, J. Biol. Chem.. 184: 745, 1950.
12. Muir, H. M., and Neuberger, A.: The biogenesis of porphyrins. 2. The origin of
the methyne carbon atoms, Biochem. J., 47: 97, 1950.
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Further evidence on the relationship of the citric acid cycle and porphyrin
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14~ Shemin, D., and Russell, C. S.: 6-Amino-levulinic acid; its role in the biosynthesis
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17. Neuberger, A., and Scott, J. J.: Aminolevulinic acid and porphyrin synthesis,
Nature (London) 172: 1093, 1953.
18. Dresel, E. I. B., and Falk, J. E.: Conversion of 6-aminolevulinic acid to por-
phobilinogen in a tissue system, Nature (London) 172: 1185, 1953.
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b-aminolevulinic acid to porphobilinogen, Biochem. J., 58: xii, 1954.
20. Gibson, K. D., Neuberger, A., and Scott, I. J.: The purification and properties
of 6-aminolevulinic acid dehydrate, Biochem. J., 61: 618, 1955.
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porphyria, Nature (London) 170: 614, 1952.
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OCR for page 73
BIOSYNTHESIS OF PORPHYRINS—SHEMIN
73
25. Falk, J. E., Dresel, E. I. B., and Rimington, C.: Porphobilinogen as a porphyrin
precursor, and interconversion of porphyrin:; in a tissue system, Nature (London)
172: 292, 1953.
26. Labbe, R., and Shemin, D.: Unpublished observation.
27. Schulman, M. P., and Rickert, D. A.: Heme synthesis in vitamin B.`; and panto-
thenic acid deficiencies, J. Biol. Chem., 226: 181, 1957.
2S. Dain, J., and Shemin, D.: Unpublished observation.
29. Weliky, I., and Shemin, D.: Unpublished observation.
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31. Winestock, C., and Shemin, D.: Unpublished observation.
32. Scott, J. J.: Synthesis of crystallizable porphobilinogen, Biochem. J., 62: 6P, 1956.
33. Corwin, A. H., and Andrews, J. S.: Studies in the pyrrole series. III. The rela-
tion of tripyrrylmethane cleavage to methene synthesis, J. Am. Chem. Soc. 59:
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34. Andrews, J. S., Corwin, A. H., and Sharp, A. G.: 1, 4, 5, 8-Tetramethyl-2, 3, 6, 7-
tetracarbethoxy-porphyrin and some derivatives, J. Am. Chem. Soc. 72: 491, 1950.
35. Abramsky, T., and Shemin, D.: Unpublished observations.
36. Hodgkin, D. C., Pickworth, S., Robertson, J. H., Trueblood, K., Piozen, R. J., and
White, J. G.: Structure of vitamin B..,, Nature 176: 325, 1955.
37. Bonnett, R., Cannon, J. R., iohnson, A. W., Sutherland, I., Todd, A. R., and Smith,
E. L.: The structure of vitamin B.., and its hexacarboxylic acid degradation
product, Nature 176: 328, 1955.
38. Shemin, D., Corcoran, J. W., Rosenblum, C., and Miller, I. M.: On the biosyn-
thesis of the porphyrin-like moiety of vitamin B~.,, Science 124: 272, 1956.
39. Corcoran, J. W., and Shemin, D.: The biosynthesis of vitamin B~.,, Biochim. et
biophys. acta (in press).
.\
Representative terms from entire chapter:
porphyrin synthesis
