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OCR for page 84
ENZYMATIC STUDIES OF
PROTOPORPHYRIN SYNTHESIS*
S. GRANICK
Introduction. The enzymes of protoporphyrin biosynthesis may be di-
vided conveniently into three groups for purposes of study:
"Active' glycine '
"Active" succinate ~ ~ Air > PBG—URO-gen ~ COPRO-gen ~ PROTO
Group I
Group II
Group III
The first group is that which may be considered to convert "active glycine"
and "active succinate" to lo-amino levulinate (bAL).i The second group con-
verts bAL to coproporphyrinogen (COPRO-gen). The third group converts
COPRO-gen to proto porphyrin. Groups I and III enzymes are connected
with Articulates; group II comprises soluble enzymes. \N7e shall consider in
turn the properties of the three groups of enzymes.)
I. ENZYMES OF THE FIRST GROUP
The first group of enzymes which cor~verts "active glycine" + "active
succinate" to bAL is a complex of enzymes and enzyme systems.' We have
been interested in this group because controls of porphyrin biosynthesis un-
doubtedly reside here. For example, one would like to know what steps
govern the differentiation of the proerythroblast into a cell in which the
predominant syntheses are those of porphyrins and globin. Marked enhance-
ment of porphyrin synthesis is also observed in certain Chlorella mutants and
in a strain of Tetrabymena toward the end of the rapid phase of growth;
here also some governing reactions toward porphyrin synthesis must be in-
volved.
To study this first group of enzymes we have followed protoporphyrin
synthesis in chicken red cells with inhibitors in order to map out what possible
enzyme systems might be involved. We were able to assign the action of
these inhibitors to the enzymes that acted between glycine and SAL, or to
the enzymes that acted between bAL and PROTO, by making tests with
glycine or with bAL as substrates. From experiments with inhibitors one
may obtain suggestive leads as to the enzymes involved. Because of the
~ These investigations were supported in part by a research grant from the Division
of Research Grants and Fellowships of the National Institutes of Health, U. S. Public
Health Service R. G. 4922.
-t SAL— 6-amino levulinate, bAL-ase enzyme which converts bAL to PBG
PBG porphobilinogen; PBG-ase enzyme which converts PBG to URO-gen,
URO — uroporphyrin; IJRO-gen — uroporphyrinogen; U D-ase enzyme which
decarboxylates URO-gen to COPRO-gen; COPRO coproporphyrin; COPRO-
gen — coproporphyrinogen; PROTO — protoporphyrin; CoASH coenzyme A;
GSH glutathione; EDTA ethylenediamine tetraacetate; Tris — tris hydroxy-
methyl aminomethane.
84
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BIOSYNTHESIS OF PROTOPORPHYRIN GRANICK 85
Glycine
Active succinate
?
fFyridoxal- P0~1
Glutamine >---^ c9AL
l Liver factors,
succinote O2
I~ CoASH ~ ~ ~ Cyt a3l FIG. 1. Some enzyme systems
Y Lipoic ~~` c /~PO4 and substances involved In the con-
TPP ~ " FADb | version of glycine to CAL.
a:-] ~elogl utarate DP~H J
Portion of ::lectron
citric acid tron sfer
c: cle system
limited time available for presentation, only the summary of the results and
the conclusions we have drawn from them are presented (fig. 1~.
On the basis of centrifugation studies some of the enzymes involved in
group I appear to reside in cell Articulates.
Cyanide and CO inhibit bAL synthesis from glycine. Therefore an oxi-
dative metabolism connected through cytochrome oxidase and the electron
transport cytochromes is inferred.
Dinitrophenol inhibits bAL synthesis. Therefore an oxidative phosphory-
lation is assumed.
Various members of the citric acid cycle when added as substrates to-
gether with glycine increase the bAL synthesis. thus confirming the involve-
ment of a citric acid cycle.
an, .
i,
~ ne electron transport system, oxidative phosphorylation, and the citric
acid cycle are known components of mitochondria. Therefore one may con-
clude that mitochondria play an active part in bAL synthesis.
The action of malonate is not simply to block succinic dehydrogenase since
SAL synthesis from glycine is inhibited equally whether succinate or a-keto-
glutarate are added as substrates. If malonate inhibited only succinic deLy-
drogenase one might have expected that SAL synthesis should not be inhibited
when glycine and a-ketoglutarate were the substrates.
Arsenite markedly inhibits bAL synthesis either when succinate or a-keto-
glutarate are added as substrates. Arsenite combines with lipoic acid and
relight block bAL synthesis in this manner. Shemin and Cumins concluded
that the citric acid cycle at succinate can back up to form succinyl-CoA.
Since bAL is not formed when either a-ketoglutarate, or succinate is added
in the presence of arsenite, it may be that the "active succinate" is a lipoic
derivative rather than a CoA derivative.
Desoxypyridoxine inhibits bAL synthesis and the inhibition may be over-
come by pyridoxal phosphate. This observation confirms the findings of
Schulman and Richer that pyridoxal phosphate is necessary for bAL
synthesis. We have also found that pyridoxal phosphate is required for SAL
synthesis in homogenates of red cells. The early nutritional studies of Wintrobe
OCR for page 86
86
PART II. BIOSYNTHESIS OF HEMOGLOBIN
and Cartwright5~6 demonstrated that the lack of vitamin Be led to the de-
velopment of an anemia in which small pale erythrocytes were produced.
V`7e may now interpret these findings as indicating that pyridoxal phosphate
has two predominant effects, namely on porphyrin and on amino acid syn-
thesis. The paleness of the red cells is due to the requirement of pyridoxal
phosphate for SAL synthesis; the smallness of the cells is due to a decreased
globin synthesis. The function of pyridoxal phosphate in bAL synthesis is
unknown. Perhaps it serves at the step which links "active succinate" to
"active glycine."
Azaserine inhibits bAL synthesis. This inhibition is overcome by glutamine
but not by pyridoxal phosphate or the aromatic amino acids. This result
suggests that glutamine may be involved in bAL synthesis. Perhaps it might be
involved in the formation of active glycine. Isonicotinic acid hydrazide also
inhibits hAL synthesis but its inhibition is overcome by pyridoxal phosphate.
Studies on hemolysates of chicken erythrocytes indicate that progressive
enhancement of protoporphyrin synthesis may be obtained by the addition of
the following substances: glycine, pyridoxal phosphate, some non-protein
fractions from pig liver. In addition, slight increases were occasionally ob-
served with inosine and with CoASH. None of the other coenzymes which
were added was found to enhance the synthesis.
Control mechanisms which may be involved in porphyrin synthesis are
suggested by the inhibitory effects of certain amino acids and keto acids.
When chicken red cells are incubated with glycine and arty one of the fol-
lowing amino acids in equimolar concentration: serine, alanine, proline or
arginine, the PROTO synthesis is decreased by 30 - 50 per cent; still greater
inhibition is found in the presence of cysteine. It is probable that the inhibition
is not by way of competition for pyridoxal phosphate since aspartate and glu-
tamate, among others, did not inhibit. On the basis of these results one
might hypothesize that when such amino acids as serine, cysteine, etc. become
limiting, a partial block to porphyrin formation might be removed. Pyruvate
and a-keto butyrate also inhibit SAL synthesis. It is suggested that these
keto acids, and perhaps ot'~e's, might compete faith a-ketoglutarate for
coenzymes.
II. ENZYMES OF THE SECOND GROUP
lathe enzymes of this group are all soluble, and serve to convert bAL to
coproporphyrinogen.7 Three colorless enzyme fractions have been separated
by zone electrophoresis on starchS at pH 7.65 in tris buffer, ionic strength
0.16 at 90mA, 5°C., and 3.1 NT/cm drop across the starch block. The enzymes
of interest migrate toward the anode, and hemoglobin migrates toward the
cathode. Figure 2 is an example of the enzyme fractions separated from a
chicken red cell supernatant solution. Red cells from three species were
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Representative terms from entire chapter:
pyridoxal phosphate
BIOSYNTHESIS OF' PROTOPORPHYR1N GRANICK
Log n~9 3
prote~n
De-
ccrboxyl(!se
P3G-ose
bAL-a<~e
2
1
0 1
08
045
-
Cm. 5 10 ~ ~
~L
~ 1
~L
J ,
20 25 30
35 ~
F`IG. 2. Zone electrophoresis on a starch blocl~ 2 cm. thick, 15 cm. wide, 45 cm. long.
A supernatant solution of a chicken red cell hemolysate was applied as a streak across
the width of the block at "Origin." The activities of eluates from 2 cm. wide seg-
ments of the starch block and their protein contents are shown at the end of the run.
Quantitative activities of bAL-ase and PBG-ase are presented (see text) and also the
qualitative distribution of UD-ase (decarboxylase).
examined: rabbit reticulocytes, chicken erytl~rocytes and cells from a case of
erythroblastosis foetalis.
hAL-ase. One fraction, bAL-ase, converts bAL to PBG. Another fractior~,
PBG-ase, converts PBG to uroporphyrinoger1 (URO-gen). The third frac-
tion, uroporphyrinogen decarboxylase (UD-ase), converts URO-gen to cop-
roporphyrinogen ~ COPRO-gen ~ ~ fig. 3 ~ . The rabbit preparation yielded
Group lI
2 `3A~ ~AI~-ase
P~G deomin~x
COOH
COOH CH2
1 1
CH2 CH2
H2~- H2C N
H
porphobilinogen
(P :ES G)
A: P ]
H2 N-H2C hl ~
pyPryl methone_
~ C 1
+ n NH3
Pr, tl Ac
AC~/ ~P~ Intermedicte Ac4~// \^Pr
deaminox N HN thene oxid tion N~ N
C Ch2 ~ product HC~ ~C
~C~ (500 my' band) ~¢ h'~Ac
Ac H2 Pr AC H Pr,
Uroporphyr~in ~ (Uro I)
Uropoophyrinogen I
(lJ~o- 9en 1)
~r' 11 AC
_~/C~_ _
I 13omerasc -' ~ H2C ~ ~ Ch2 ~ _
~C;
Pr' H2 Pr,
Uro-gen ]lI
ldecanboxylase
C2 C~3
~ ~ N
H3C
88
PART II. BIOSYNTHESIS OF HEMOGLOBIN
eluates from the starch which possessed tile highest activities. The maximum
bAL-ase activity was 0.8 ,uMoles PBG formed per hour per ma. protein.
The maximum PBG-ase activity was 0.44 Moles PBG decrease per hour
per ma. protein. The maximum UD-ase activity was 0.009 Moles COPRO-
gen formed per hour per ma. protein from URO-gen as substrate. The
relative rates of migration toward the anode of the rabbit enzymes was
bAL-ase > UD-ase > PBG-ase; of the chicken enzymes bAL-ase ~ PBG-
ase > UD-ase; and of the human, UD-ase > PBG-ase > DAL-ase.
COON COOH
1 1
COOH CH2 COOH CH2
I A I Ch2 CH2
C OCHER --CO ~ ~
H2N-H2C-CO B ~ H2 H~N-CH2~ iLH
NH2 N
FIG. 4. Hypothesis of action of bAL-ase at the active site.
The properties of bAL-ase have been studied kinetically and with inhibitors.
A hypothesis of the action of bAL-ase is presented in figure 4. The kinetic
studies may be interpreted as indicating that one SAL is held more firmly
than the second SAL, as judged from the slope of the curve of rate vs. log
bAL concentration at low (10-4M) and at higher concentrations. From in-
hibitor studies it is inferred that the attachment of bAL to the active site is
relatively strong at the carbonyl group, relatively weak at the carboxyl group
and negligible at the NH group. After both bAL molecules have been ad-
sorbed on the active site, a l~etimine condensation should occur readily and
spontaneously at B since pyrazine formation between two bAL molecules is
known to occur readily.9 Ketimine formation occurs only with unionized
NEIL groups; the decrease in bAL-ase activity below pH 6 may be due in
part to the decrease in the unionized NH groups of SAL. (In addition there
is a temperature-dependent denaturation below pH 6~.
It is necessary to postulate some mechanism for the activation of the hy-
drogen at C. A strong positive charge in the neighborhood of this atom
might serve for activation. Such a positive charge might be imparted by a di-
or trivalent metal ion; however no evidence for a metal ion activation has
been obtained. (Studies of the inhibition of bAL-ase activity by EDTA in-
dicate a reversible inactivation of a structurally-modified form of CAL-ase.)
Thus the formation of the two bonds are interpreted to be due to a spon-
taneous ketimine condensation at B and an enzyme activated condensation at A.
PBG-ase. When a specific enzyme fraction prepared by zone electro-
phoresis acts on PBG' the colorless compound URO-gen isomer type III is
BIOSYNTHESIS OF PROTOPORPHYRIN GRANICK 89
formed. However when this fraction is heated for an hour at 55°C. this
heated fraction now converts PBG to URO-gen isomer type I. The result
of the heat treatment is similar to that found in Chlorella.~°
The erect of heat treatment suggests that the PBG-ase fraction consists
(_7
r
ot two enzymes. ()ne enzyme, "deaminase," may functior~ to form methane
bonds between adjacent PBG molecules with the concomitant elimination of
NH; (fig. 3~. If this action were to continue, the symmetrical URO-gen
I should result, which is not further changed by the PBG-ase preparation.
The second enzyme, "isomerase," would act on a product of the deaminase
to convert it to URO-gen III. The isomerase enzyme is probably the one
that is readily destroyed by heat.
With spinach extracts Bogoradi~ has been able to separate two activities:
one which results in the conversion of PBG to URO I, and the other which
appears to act on some intermediate stage of the condensation to convert the
intermediate to URO III.
In the disease of chronic porphyria)'' which is due to a recessive gene,
large amounts of URO I and COPRO I are excreted. :From the above con-
siderations it is suggested that this metabolic lesion may be due to a diminu-
tion in the active "isomerase" enzyme.
Uroporphyrinogen decarboxylase (UD-ase). When a specific fraction
prepared by zone electrophoresis acts on URO-gen then COPRO-gen is
formed.~3 The enzyme fraction is incubated anaerobically in the presence of
0.005M GSH and URO-gen III (prepared from URO by reduction with
sodium amalgam) at pH 6.8. at 38°C. in the dark. The results of the decar-
boxylation are shown in figure 5. During the reaction the intermediate 7-, 6-
and 5-carboxylic porphyrinogens remained at a low, roughly steady-state con-
centration while the 4-carboxylic compound COPRO-gen continued to accum-
ulate. The porphyrinogens were quantitatively assayed by titration with
iodine. The oxidation of porphyrinogens with iodine and their autoxidative
sensitivity to light were reported by Watsoni4 and are confirmed by the
present quantitative studies.
~ ~ c: Q c' c'
.
.
o ~ ~ ~
o 8 ~ ~ ~
UPO 6X10-5 6X10-5 6X10 5M 6X10-611
1hI? 4 hr. 23 Ire. 1 hr. 23 hr. NOenZYme~
23 hr.
6X10-6~ 6X10-5 ~ CONDO
FIG. 5. Action of UD-ase on URO-gen at indicated concentrations to form decar-
boxylated porphyrinogens. The porphyrinogens were oxidized with iodine and the
porphyrins separated by paper chromatography in a lutidine-aqueous NH3 system.
9o
PART II. BIOSYNTHESIS OF HEMOGLOBIN
The following properties of UD-ase from rabbit reticulocytes have been
determined. The enzyme is an -SH enzyme whose activity is decreased by
-SH-binding reagents and protected by GSTI. The apparent Michaelis con-
stant for the enzyme is < 10-5M, and the optimum pH is 6.8. The enzyme
decarboxylates all the four URO-gen isomers; the relative order of efficiency
is III > IV > II -- I. Isomer III is decarboxylated about twice as fast as I.
Only the fully reduced porphyrins and not the intermediate states of oxi-
dation (pyrromethenes) are acted upon by this enzyme fraction. This con-
clusion is based on kinetic studies and on the fact that the presence of either
sulfite or dithionite, which combine with the pyrromethenes, does not affect
the rate of decarboxylation. Leo isomerization of the porphyrinogens occurs
either on reduction with sodium amalgam, on incubation with the enzyme
fraction, or on oxidation with iodine. The enzyme fraction does not de-
carboxylate bAL, PBG, URO or indole acetic acid.
III. ENZYMES OF THE THIRD GROUP
The conversion of COPRO-gen to PROTO is brought about by cell
Articulates of the red cell and of Euglena but the intermediate steps are
still unknown. Of appears to be necessary, but the reaction is not cyanide
sensitive; therefore cytochrome oxidase is probably not involved. COPRO is
not attacked but only the fully reduced COPRO-gen. Comparison of the
behavior of COPRO and PROTO on reduction indicates that reduced
PROTO is more readily autoxidizable than reduced COPRO under the
. .
same cone ltlons.
One interesting problem in connection with studies on porphyrin biosyn-
thesis is the localization of this biosynthetic chain in the cell. Two proto-
plasmic organelles which contain porphyrin compounds are the mitochondria
with their cytochromes, and the chloroplasts which, in addition to chlorophyll,
also possess cytochromes. The fact that bAL synthesis from glycine requires
mitochondrial enzymes and that cell Articulates are involved in the con-
version of COPRO-gen to PROTO suggests that the synthesis of PROTO
might be a function of such organelles. In this regard it would be interesting
to know whether the soluble enzymes, which convert bAL to COPRO-gen,
are also concentrated in these bodies.
Another interesting problem is determining the control mechanisms and
protective devices for maintenance of porphyrin biosynthesis. Suggestive of
some control is the inhibitory action on SAL synthesis of certain amino and
keto acids. For maintenance, an obvious mechanism is the high GSH content
of the erythrocyte. Not only does GSH serve to maintain the activity of the
-SH enzymes (bAL-ase and UD-ase) but GSH also serves to decrease the
autoxidation of the porphyrinogens. It has also been observed that the high
protein content of red cells would serve to stabilize bAL-ase, which is un-
BIOSYNTHESIS OF PROTOPORPHYRIN—GRANICK 91
stable in dilute solutions, and would also serve to protect PBG from under-
going condensation and decompositior~ reactions.
IV. SUMMARY
The enzyme systems which take part in the conversion of glycir~e to PAL
include the citric acid cycle, the electron transport system, and oxidative
phosphorylation. These systems are presumably localized in mitochondria.
In addition to the compounds required for the above systems, studies with
inhibitors and red cell hemolysates implicate the following compounds in
this synthesis, namely: pyridoxal phosphate, glutamine, inosine, and some
as yet unidentified non-protein liver factors (fig. 1~.
The conversion of bAL to COPRO-gen has been shown to take place via
three soluble enzyme fractions which have been separated from red cells by
zone electrophoresis (fig. 2~. One fraction unites two bAL molecules together
to form PBG (fig. 3~. Studies on the mechanism of this reaction are reported
(fig. 4~. Another fraction appears to contain two enzymes, one of which, a
deaminase, is hypothesized to unite PBG molecules to form a pyrro-methane
type intermediate which is then acted upon by another enzyme, isomerase, to
form URO-gen III. The third fraction contorts a decarboxylase which acts
on URO-gen to decarboxylate the acetic side chains to methyl groups, to
result in the formation of COPRO-gen (fig. 5~.
The conversion of COPRO-gen to PRC)TO requires cell Articulates arid
Or. It is not poisoned by cyanide.
REFEREN CES
1. For a review of the pertinent literature see: Wolstenholme, G. E. W., and Millar,
E. C. P., eds.: Ciba Foundation Symposium on Porphyrin Biosynthesis and
Metabolism. London, Churchill; Boston, Little, Brown and Co., 1955.
2. Granick, S.: Enzymes in the early steps of porphyrin biosynthesis to delta-amino
levulinate. In preparation.
3. Shemin, D., and Kumin, S.: The mechanism of porphyrin formation. The forma-
tion of a succinyl intermediate from succinate, J. Biol. Chem. 198: 827, 1952.
4. Schulman, M. P., and Richert, D. A.: An effect of pyridoxal-5-phosphate in vitro
on heme synthesis and CO2 production from glycine-2-C-14, J. Am. Chem. Soc.
77: 6402, (December 5) 1955.
5. Wintrobe, M. M.: Factors and mechanisms in the production of red corpuscles,
Harvey Lectures 45: 87, 1949-50, Academic Press, New York.
6. Cartwright, G. E., and Wintrobe, M. M.: Studies on free erythrocyte protopor-
phyrin, plasma copper, and plasma iron in normal and pyridoxine-deficient
swine, J. Biol. Chem. 172: 557, 1948.
7. Granick, S., and Manzerall, D.: Enzymes that convert delta-amino levulinate to
coproporphyrinogen. In preparation.
8. Kunkel, H. G.: Zone electrophoresis, Methods of Biochem. Analyses 1: 141, 1954.
9. Gibson, K. D., Neuberger, A., and Scott, J. J.: The purification and properties of
delta-aminolaevulic acid dehydrate, Biochem. J. 61: 618, 1955.
10. Bogorad, L., and Granick, S.: The enzymatic synthesis of porphyrins from por-
phobilinogen, Proc. Nat. Acad. Sci. 39: 1176, 1953.
