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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

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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.

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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.

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BIOSYNTHESIS OF PORPHYRINSSHEMIN 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

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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 CH2NH2 Ac P P Ac FIG. 4.A mechanism of porphyrin formation from the monopyrrole. AcAcetic acid side chain; P Propionic acid side chain; ~ a-carbon atom of glycine and 6-carbon atom of 6-aminolevulinic acid.

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BIOSYNTHESIS OF POF(PlIVRINSSHEMIN 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.

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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. 13. Wriston, J. C., Lack, L., and Shemin, D.: The mechanism of porphyrin formation. Further evidence on the relationship of the citric acid cycle and porphyrin formation, J. Biol. Chem., 215: 603, 1955. 14~ Shemin, D., and Russell, C. S.: 6-Amino-levulinic acid; its role in the biosynthesis of porphyrins and purines, J. Am. Chem. Soc., 75: 4873, 1953. 15. Shemin, D., Russell, C. S., and Abramsky, T.: The succinate-glycine cycle. I. The mechanism of pyrrole synthesis, l. Biol. Chem., 215: 613, 1955. 1 6. SchiRmann, E., and Shemin, D.: Further studies on the utilization of b-amino- levulinic acid for porphyrin synthesis, J. Biol. Chem., 225: 623, 1957. 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. 19. Gibson, K. D., and Neuberger, A., and Scott, J. l.: The enzymic conversion of 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. 21. Schmid, R., and Shemin, D.: The enzymatic formation of porphobilinogen from b-aminolevulinic acid and its conversion to protoporphyrin, I. Am. Chem. Soc., 77: 506, 1955. 22. Granick, S.: Enzymatic conversion of 6-aminolevulinic acid to porphobilinogen, Science, 120: 1105, 1954. 23. Westall, R. G.: Isolation of porphobilinogen from urine of a patient with acute porphyria, Nature (London) 170: 614, 1952. 24. Cookson, G. H., and Rimington, C.: Porphobilinogen. Chemical constitution. Nature (London) 171: 875, 1953.

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BIOSYNTHESIS OF PORPHYRINSSHEMIN 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. 30. Levenberg, B., Melnick, I., and Buchanan, J. M.: Biosynthesis of purines. XV. The effect of aza-L-serine and 6-diazo-5-oxo-L-norleucine on inosinic acid biosynthesis de Volvo, J. Biol. Chem. 225: 163, 1957. 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: 1973, 1937. 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). .\