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THE ENZYMATIC SYNTHESIS OF UROPORPHYRINOGENS FROM PORPHOBILINOGEN* LAWREN CE B O GORAD You have heard ~ report on the elegant work of Dr. Shemin and his colleagues relating b-aminolevulinic acid to the Krebs cycle on the one hand and to porphyrin biosynthesis on the other. Also, several laboratories have reportedi~'~3 the isolation of an enzyme which catalyzes the condensation of two molecules of b-aminolevulinic acid to form one of porphobilinogen COOH COOH ,. I CH2 CH, COOH 1 CHEF C O CON He - DAL Cow Ha\ CHEF 1 a' ,—CHINO \ N ~ PORPHOBILINOGEN FrG. 1. Structural formulae for b-aminolevulinic acid and porpho- bilinogen. (PBG) (fig. 1~. The utilization of PBG in the biosynthesis of porphyrins, including protoporphyrin IX, has been demonstrated using a number of different sources of enzymes;4~5~6 table I shows the nature of the porphyrins recovered after the incubation of PBG with a frozen and thawed preparation of Chlorella cells.4 This work supported a great deal of earlier evidence (e.~., reference 7) which suggested that protoporphyrin IX is derived from ~ - ~ 7 - ~ 0= ~ ~ ~ ~ an octacarboxyl~c tetrapyrrole and furthermore Indicated that all the naturally- occurring porphyrins originate by the condensation of four molecules of the same pyrrole, i.e. porphobilinogen. The present report deals primarily with investigations of enzymes which catalyze steps in the synthesis of uroporphyrins, and their immediate pre- cursors, from PBG. There are four possible uroporphyrin isomers but only two, uroporphyrin I and uroporphyrin III (fig. 2), are known to occur in nature. Uroporphyrin I can be visualized as being formed by the linear con- densation of four PBG molecules followed by ring closure and oxidation, ¢' this work was supported by grants frorr the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, and from the National Science Foundation. It was also supported in part by the Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago. 74
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BIOSYNTHESIS OF UROPORPHYRINOGENS—BOGORAD 75 TABLE I PCRPHYRINS PRODUCED DURING THE INCUBATION OF PBG AND FROZEN AND THAWED CHLORELLA PREPARATIONS4 Porphyrins Fraction Present of Total Porphyrin Synthesized Aqueous Acetone-HC1 (2N HC1) Uroporphyrin 2, 3, 4, 5, 6, 7* 2,4, 5* = Protoporphyrin 285fo 52% 6% 1470 * Numbers refer to number of carboxyl groups per molecule. Underlined porphyrins present in greatest abundance. P H AC AC Am// up HI NH H; AC ~N /~AC P H P Uroporphyrin 111 AC = -CH2-COOH P =—CH2- CH2- COOH AC W P N N HC~\ BACH IN N=: P in\ ILIAC Ac H p U~oporphyrin I FIG. 2. Structural formu- lae for uroporphyrin I and III. but a tetrapyrrole with this arrangement of propionic and acetic acid side chains is unsuitable as an intermediate in the synthesis of protoporphyrin IX, unless a mechanism for shifting these substituents exists. On the other hand! the arrangement of side chains as on uroporphyrin III would fit the re- quirements of a precursor of protoporphyrin IX. Thus, the problem with respect to the biosynthesis of protoporphyrin IX is not merely to make a tetrapyrrole from PBG but to make tile proper one. In the initial studies of individual enzymes in this phase of porphyrin bio- synthesis,S~9 aqueous extracts of acetone powders of spinach leaf tissue were subjected to ammonium sulfate fractionation. The course of PBG con- sumption and the appearance of uroporphyrin in the presence of one fraction are shown in figure 3. Melting point determinations and paper chrom- atographyi° have shown that the porphyrin produced in such a reaction is about 99/ uroporphyrTr~ I. Further purification of this enzyme, porphobilin- oger1 deaminase, in this fraction was accomplished by mild heat treatment and zone electrophoresis. Purified preparations differ from the cruder ones in that, when they are used to catalyze the reaction, the appearance of porphy- rin lags far behind the consumption of PBG. As is shown in figure 4, using one such preparation, at the time of near exhaustion of the substrate less than
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76 PART II. BIOSYNTHESIS OF HEMOGLOBIN 0.5 0.4 0.3 0.2 O. 1 \ PBG / ~ IBM Porphyrin x 4 30C 200 - <, a' 100 O 60 120 180 240 3< )0 minutes Ml NUTES 1 :~ PI - 60 0 :r At, , _ _. FIG. 3. (left) The course of the consumption of PBG and the appearance of uro- porphyrin I. The reaction shown here was catalyzed by PBG deaminase in a 40-50C/c ammonium sulfate fraction of an aqueous extract of spinach leaf tissue. FIG. 4. (right) Lag in production of porphyrin when using purified preparation of PBG deaminase. Compare with faster reaction shown in figure 3. 15% of the PBG consumed can be accounted for as porphyrin, while the maximum final yields of porphyrin have, in some experiments, approached l OO 'Jo . It was found ' ' later that, using either the crude or more purified preparations, anaerobic conditions have no effect on the rate of PBG con- sumption but the appearance of porphyrin is completely suppressed. During the consumption of PBG, either aerobically or anaerobically, one mole of ammonia is released for each mole of PBG which disappears. These data suggest that no oxidative step, e.g. the formation of a pyrrole aldehyde, need occur in the course of the condensation of PBG molecules to form the color- less intermediate. The following observations make it clear that this colorless material is uroporphyrinogen I, an interesting and important intermediate in porphyrin biosynthesis. It can be oxidized to uroporphyrin I rapidly by iodine, or, more slowly, by aerobic incubation, or an enzymatic oxidation can be accomplished by the addition of a small amount of crude deaminase preparation. (Thus, the crude deaminase preparations from spinach leaf tissue appear to contain two enzymes which are active in porphyrin biosynthesis, porphobilinogen deaminase and this oxidase, the specificity of which has not yet been determined). From the point of view of subsequent steps in porphyrin biosynthesis it is especially interesting that the colorless product of the deaminase reaction can serve as a substrate for enzymes present in frozen and thawed preparations of Chlorella which catalyze the synthesis of porphyrins with fewer than eight
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BIOSYNTHESIS OF UROPORPHYRINOGENS BOGORAD 77 carboxyl groups per molecule. Since these enzymes cannot use uroporphyrin I as a substrate, it is obvious that this colorless material is not oxidized to uro- porphyrin I prior to its being acted upon by these enzymes. The data in TABLE II PORPHYRIN CC~VERSIONS BY FROZEN AND THAWED CHLORELLA PREPARATIONS*; Porphyrin Substrate Recovered ,uM x 4 Nature of porphyrins recovered* % % Uroporphyrin other soluble Porphyrins 5.76 AM PBG-equiv- alents from PBG + PBG-D incubation 4.21 18.1 81.9 5.52 AM PBG ~ 4.02 ~ 22.0 1 78.01 3.49 AM PBG equiv- alents as uroporphy- rinogen I 2.29 1 24.5 75.5 ~ 2.55 EM PBG equiv- alents as uroporphy- . ran I 2.14 1 100.0 0.0 * Frozen and thawed Chlorella preparation incubated with substrate anaerobically, then aerobically to oxidize porphyrinogens. -I Mostly coproporphyrin but includes traces of porphyrins with 2, 3, and .o - COOH groups/ molecule. Table II show that this colorless material and reduced uroporphyrin I, i.e., uroporphyrinogen I, serve equally well as substrates for these enzymes. These data, and others, show that the colorless product of the deaminase reaction is uroporphyrinogen I ~ fig. 5 ~ . Thus, judging from the fact that the product is the completely symmetrical uroporphyrinogen I, PBG deaminase appears to catalyze the linear con- densation of PBG molecules and ring closure of the tetrapyrrole but, as pointed out above, the arrangement of side chains on this isomer probably renders it valueless as an intermediate in the biosynthesis of protoporphyrin IX. Another enzyme, uroporphyrinogen isomerase, which participates in the synthesis of uroporphyrinogen III from PBG, has been partially purified from aqueous extracts of wheat germ. Crude aqueous extracts catalyze the consumption of PBG and the appearance of uroporphyrin III, sometimes mixed with uroporphyrin I, but two ammonium sulfate fractions are of par- ticular interest. One of these, Fraction B. catalyzes the consumption of PBG and the appearance of uroporphyrin III, sometimes mixed with uroporphyrin
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78 PART II. BIOSYNTHESIS OF HEMOGLOBIN COOH COOH COO H COOH 1 1 1 1 COOH CH2 COOH CH2 COOH CH2 COOH CH2 CH2 CH2 CH2 CH2 CH2 C ~ 2 CH2 CH 2 OWL cili ~~ H: N N N H CH H H HCH HCH ~ ~ HCH \:H ~ ~ HI CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 COOH CH2 CH2 COOH CH2 COOH CH2 COOH COOH COO H COOH COOH lJROPoRPHyRlNoGEN m UROPORPHYRINOGEN I FIG. 5.—Structural formulae for uroporphyrin I and III. I. After aging for two days at 4°C. or heating for 15 minutes at 55°C. the capacity of this fraction for catalyzing PBG consumption is unaltered, but only uroporphyrin I is produced. This suggested that at least two enzymes might be involved in the biosynthesis of uroporphyrin III and that one enzyme is more heat labile than the other. Fraction C, from wheat germ, is of greater interest. Preparations of this fraction vary in their capacity to catalyze the consumption of PBG when incubated alone w-ith this pyrrole; some preparations are slightly active, while others possess no measurable activity. If, however, a preparation which is inactive by these standards is incubated with PBG deaminase prepared from spinach leaf tissue, as well as w-ith PBG, the substrate is consumed at a rate commensurate with the concentration of the deaminase but the III, rather than the I isomer of uroporphyrin, is produced. Up to 100/ of the PBG consumed has been accounted for as porphyrin in some of these experiments. The product has been characterized as uroporphyrin III by paper chromatography10 and melting point determinations of the octamethyl ester and by similar analyses of the methyl ester of coproporphyrin produced by partial decarboxylation of the uroporphyrin. ~ Pooled material from 5 experiments was estimated to contain 85-90 t70 uroporphyrin III and 10- 15~/o of the I isomer). As in the case of the enzymatic production of uro- porphyrin I, the activity of the deaminase-isomerase system is not impaired by anaerobiosis but, a~ain, the uroporphyrino~en, rather than uroporphyrin, accumulates. The uroporphyrinogen III so produced can be utilized by frozen and thawed Chlorella preparations as a substrate for the synthesis of a number of porphyrins, including protoporphyrin. (Also see reference no. 12~. Thus we are clearly on the right track for protoporphyrin biosynthesis.
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BIOSYNTHESIS OF UROPORPHYRINOGENS BOGORAD 79 In order to determine whether both the deaminase and the isomerase need be present simultaneously for uroporphyrinogen III biosynthesis, or whether these enzymes can act serially on PBG, the following experiments were performed. In one group of experiments PBG was incubated with the deaminase anaerobically, and then, at the time of exhaustion of the substrate, uroporphyrinogen isomerase was added. This had no effect ore the nature of the product, i.e. uroporphyrin I was finally recovered. So the possibility of the shifting of acetic and propionic acid side chains as a mechanism of iso- merization appears to be as unlikely as would have been predicted. In another series of e~cperimer~ts it was found that the exposure of PBG to the isomerase first and then to PBG deaminase is an equally ineffective means of producing uroporphyrin III. Experiments were performed in which PBG was incubated with uroporphyrinogen isomerase for three hours at 37 °C. When the isomerase was inactivated by heating the solution at 5 5 ° C. for 30 minutes, PBG deaminase was added, and the solution was incubated, uroporphyrin I was recovered (fig. 6, no. 14~. Uroporphyrin I was also recovered where uroporphyrinoger~ isomerase was never included in the incubation mixture, (fig. 6, no. 18) or when the isomerase was inactivated by- heating before PBG was added to the solution (fig. 6, no. 18~. OR the other hand, as already described, when urops~rphyrinogen isomerase, and PBG are ir~cubated all together (fig. 6, no. 17~' uroporphyrin III is produced. Cooksor~ and Rimingtoni4 have suggested that the switching of the amino- methyl group from one a-position to the other of PBG might be involved ire, at least, the non-enzymatic synthesis of uroporphyrinogen III from PBG. Then one molecule of isoPBG could condense with three molecules of PBG, or a linear tripyrrole produced from three molecules of PBG, to make uro- porphyrinogen III. The results of the experiment described above clearly exclude the possibility that the isomerase might act to catalyze such a shift when PBG is incubated alone with it. (Such a shift would not be reflected . . O W IOB O . O ~ O O O O ~ ~ ~ ,°2. ~ ~ ,'. " ~ 13 14 ~16 17 18 Ui U ~ FIG. 6. Paper chromato- gram (Falk and Benson methodic ) of methyl esters from preincubation experi- ment. ( See text for treat- ments. ) Dotted lines show position of pigments at the beginning of the second de- velopment; solid lines show final positions. UI = uro- porphyrin I marker; IT III uroporphyrin III marker.
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80 PART II. BIOSYNTHESIS OF HEMOGLOBIN in a change in the concentration of PBG using the Ehrlich p-dimethylamino- benzaldehyde assay). This is particularly obvious from the observation that uroporphyrin I is produced when the isomerase is inactivated after the first incubation with PBG but prior to the introduction of PBG deaminase (fig. 6, no. 14), while uroporphyrin III is recovered if the inactivation step is omitted ~ fig. 6, no. 13 ~ . Thus, uroporphyrinogen isomerase appears to be incapable of contributing to the synthesis of uroporphyrinogen I I I in the absence of the deaminase. The requirement for the presence, simultaneously, of PBG deaminase, uroporphyrinogen isomerase, and PBG for the enzymatic synthesis of uroporphyrinogen III is apparent. The next question which arises is: Is there any direct interaction between PBG and uroporphyrinogen isomerase ? At present only indirect evidence from kinetic studies is available. As I have indicated, the rate of consumption of PBG in the presence of the deaminase-isomerase system is not measurably different from the rate in the absence of the isomerase. This is true at relatively high substrate levels (100-400 ~g./ml.~; however, kinetic data reveal marked differences in the two reactions. Table III shows that there is a difference in the apparent TABLE III vm PBG-Deaminase + PBG 0.076 ~M/ml; PBG-Deaminase + PBG ~ Uroporphyrinogen Isomerase Ks 7.2 x 10 - 5 M/L. 0.125 ~M/ml. 10.4 x 10 - ~ M/L. Michealis-~:enten constant and that maximum reaction velocity is attained at a higher substrate concentration in the presence of both enzymes than when only the deaminase is present. (The uroporphyrinogen isomerase prep- aration used in these experiments failed to catalyze any measurable change in PBG concentration under these conditions when incubated alone with this pyrrole at any of the substrate concentrations studied). These data are compatible with the conclusion that there is a direct interaction between the isomerase and PBG at some point in the course of the synthesis of uropor- phyrinogen III and that this enzyme requires two substrates: PBG and some product of the action of PBG deaminase of PBG short of a cyclized tetra- pyrrole, presumably a linear di- or tripyrrole. Finally, what is the mechanism by which uroporphyrinogen III is assembled from PBG enzymatically? Dr. Shemin has introduced a scheme involving a tripyrrylmethane intermediate. This proposal13 and another one involving a "T" tetrapyrrylmethane intermediates appear to be in conflict with the observation that uroporphyrinogen III can be synthesized from PBG enzy- matically under anaerobic conditions, since the production of the "T" struc- ture from a di- or tripyrrylmethane and PBG would require an oxidation.; ~ See further qualifications and comment in Addendum at the end of this paper.
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BIOSYNTHESIS OF UROPORPHYRIi\OGENS :~3OGOR.ND 8 The proposal of Cookson and Kimingtoni~t has already been mentioned. The present observations on the deaminase-isomerase system neither confirm nor contradict the principles of their group transfer hypothesis but do sug- gest that, if such a mechanism is involved in the enzymatic synthesis of uro- 1 o~-phyrinogcn III, the transfer of the a-substituent must, most likely, occur when both substrates of uroporphyrinogen isomerase ~ PBG and a linear polypyrrole formed from PBG by the action of PBG deaminase ~ are on the surface of the enzyme. This conclusion is supported by studies on the effects of certain PBG analogues on this system. The final cyclization might then be catalyzed by the isomerase, PBG deaminase, or by another enzyme which may be present in the wheat germ preparations, for it should be pointed out that, while the purified deaminase preparations most probably certain only one enzyme which is active in nornhvrin biosynthesis. i.e. PBG ~ . 1 1 ~ ~ deam~nase, wheat germ ~ Action A; may contain, in addition to uroporp~y- rinogen isomerase, other enzymes which are active in uronorobvrin III svn- J ~ 1 , thesis. It is also possible that "uroporphyrinogen isomerase" is, in fact, group of enzymes. The lability of the isomerase and technical problems in the ready determination of the relative proportions of uroporphyrins I and III in mixtures have thus far discouraged attendants to purify this material further. Summary: This report describes two enzymes involved in the synthesis of porphyrinogens f rom porphobilinogen. One of these, porphobilinogen deaminase, catalyzes the consumption of PBG and the production, from it, of uroporphyrinogen I. The second, uroporphyrinogen isomerase, appears to be unable to catalyze any modification of PBG when incubated with it alone, but, when this enzyme is incubated with PBG and PBG deaminase, uro- porphyrinogen III is produced. These enzymatic products can serve as sub- strates for enzymes present in frozen and thawed preparations of Chlorella which catalyze the synthesis of porphyrins with fewer than eight carboxyl groups per molecule. ADDENDUMS Two hvdrogens, one from the "free a-position" of PBG and one from the methane bridge of the di- or tripyrrylmethane, must be removed in order to form any of the "T" structures which have been suggested4~~3 as intermediates in the enzymatic synthesis of uroporphyrinogen III. The next step in such schemes, howsoever, involves a cleavage of the "T" intermediate by the ad- dition of one hydrogen to each of the products. Thus, there is no net oxi- dation, and an enzyme which could accept two hydrogens and then give them up to the final products of the reaction could act anaerobically; there- fore, the observation that uroporphyrinogen III can be formed enzymatically * This Addendum and its accompanying figure were not presented at the Con- ference but were later submitted as relevant "afterthoughts," particularly with re- gard to the "T" structures discussed in the paper.
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89 PART II. BIOSYNTHESIS OF HEMOGLOBIN UROPORPHYRINOGEN I ~ 2NH~ P BG - Dl+ PBG H zN H 2C ~ C ~ C 13 H ~ 2 N H 3 -If+ Enz (UG-Ist) /~2NH2C9lC|Enz Hl:;3~C:H If- PBG H H C H \Enz+H2NH2C~CiNjlCN 2 ~Hl3 \ ~ ~ P = CH2 CH2 COOH PBG-D = PORPHOBILINOGEN DEAM I NASE = CH2 COO H -~H2NH2C(N\:C ; ~CH2NH2 ,l(PBG- D:) US - Is = UROPORPHYRINOGEN I SOMERASE URO POR PHYR I NOGEN m ~ 2 NH3 FIG. 7. Formula diagram for possible mechanism of biosynthesis of uroporphyrin ogen III f rom PBG. from PBG under strictly anaerobic conditions does not necessarily exclude the possibility of "T" pyrrylmethanes as intermediates. Another possible mechanism of uroporphyrinogen III biosynthesis from PBG could involve an enzyme with transpyrrylase activity (fig. 7 ). According to this hypothesis, first a linear tripyrrylmethane would be formed through the action of PBG deaminase on PBG. Then a transpyrrylizing enzyme would act to catalyze the exchange of a molecule of PBG for a dipyrrolic segment of the tripyrrole. This would then leave, in the reaction mixture, two dis- similar dipyrrylmethanes; the condensation of one molecule of each of these two dipyrrylmethanes would lead to the formation of uroporphyrinogen III. One of the condensations required is of the sort which PBG deaminase ap- pears to catalyze; the other condensation might possibly also be catalyzable by PBG deaminase, but another enzyme might be required. If this is the mechanism of uroporphyrinogen III biosynthesis, uroporphyrinogen isomerase acts as a transpyrrylase. Results obtained in experiments w ith PBG ana- logues argue against a dipyrrylmethane as the substrate for the transpyrrylase postulated here.
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BIOSYNTHESIS OF UROPORPHYRINOGENS BOGORAD 83 REFERENCES 1. Granick, S.: Enzymatic conversion of 6-aminolevulinic acid to porphobilinogen, Science 120: 1105, 1954. Schmid, R., and Shemin, D.: The enzymatic formation of Porphobilinogen from 6-aminolevulinic acid and its conversion to protoporphyrin. J. Amer. Chem. Soc. 77: 506, 1956. 3. Gibson, K. D., Neuberger, A., and Scott, J. J.: The enzymic conversion of 6-amino- levulinic acid to porphobilinogen, Biochem. J. 58: xii, 1954. 4. Bogorad, L., and Granick, S.: The enzymatic synthesis of porphyrins from porphobilinogen, Proc. Natl. Acad. Sci. (U.S.) 39: 1176, 1953. 5. Foals, J. E., Dresel, E. J. B., and Rimington, C.: Porphobilinogen as a porphyrin precursor, and interconversion of porphyrins, in a tissue system, Nature 17~?: 292, 1953. 6. Schulman, M. P.: Enzymatic synthesis of Porphobilinogen from b-aminolevulinic acid and its conversion to porphyrins, Fed. Proc. 14: 277, 1955. 7. Granick, S.: The metabolism of heme and chlorophyll. In "Chem. Pathways of Metabolism" Vol. I, D. M. Greenberg, ea., Acad. Press, N. Y., 1954. 8. Bogorad, L.: Enzymatic synthesis of uroporphyrin, Fed. Proc. 14: 184, 1955. 9. Bogorad, L.: Intermediates in the biosynthesis of porphyrins from porphobilinogen, Science 121: 878, 1955. 10. Falk, J. E., and Benson, A.: Separation of uroporphyrin esters I and III by paper chromatography, Biochem. J. s5 101, 1953. 11. Bogorad, L.: The enzymatic synthesis of uroporphyrin III, Pi. Physiol. 30: xiv. 1955. 12. Neve, R. A., Labbe, R. F., and Aldrich, R. A.: Reduced uroporphyrin III in the biosynthesis of heme, J. Amer. Chem. Soc. 78: 691, 1956. 13. Shemin, D., Russel, C. S., and Abramsky, T.: The succinate-glycine cycle. I. The mechanism of pyrrole synthesis, J. Biol. Chem. ~?15: 613, 1955. 14. Cookson, G. fI., and Rimington, C.: Porphobilinogen, Biochem. J. 57: 476, 1954.
Representative terms from entire chapter: