Cover Image

Not for Sale



View/Hide Left Panel
Click for next page ( 85


The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement



Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.

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 > PBGURO-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

OCR for page 84
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 84
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, 5C., 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

OCR for page 84
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 OCR for page 84
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

OCR for page 84
BIOSYNTHESIS OF PROTOPORPHYRIN GRANICK 89 formed. However when this fraction is heated for an hour at 55C. 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 38C. 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.

OCR for page 84
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-

OCR for page 84
BIOSYNTHESIS OF PROTOPORPHYRINGRANICK 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.