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suits suggesting that the metagon is m-RNA of M genes. Failure of the metagonto hybridize with DNA from Didinium indicates that this organism has no genecomparable to the M genes of Paramecium. Attempts to account for all of thefacts by assuming that the metagon increases only by replication or only by produc-tion from host genes meet with difficulties. The metagon appears to replicate likean RNA virus in Didinium and to arise as the m-RNA of M genes in Paramecium.Its possible relation to Wright's concept of the plasmagene and to the origin ofRNA tumor viruses is recognized.

Note added in proof: A full and clear summary of the background work on the metagon hasjust appeared: Beale, G., in Cellular Control Mechanisms and Cancer, ed. P. Emmelot and0. Muhilbock (Amsterdam: Elsevier Publ. Co., 1964), pp. 8-18.

* Contribution no. 747 from the Department of Zoology, Indiana University. The experimentswith metagons and mu were carried out by Ian Gibson. Aided by grant to T. M. SonnebornAT(11-1)-235-10 of the Atomic Energy Commission.

t Postdoctoral fellow, Public Health Service genetics training grant. Present address:Department of Zoology, University of Washington, Seattle 5.

1 Sonneborn, T. M., Advan. Virus Res., 6, 229 (1959).2 Gibson, I., and G. H. Beale, Genet. Res., 2, 82 (1961).3 Ibid., 3, 24 (1962).4 Ibid., 5, 85 (1964).6 Sonneborm, T. M., in preparation.6 Gibson, I., in preparation.7 Gibson, I., Proc. Roy. Soc. (London), in press.8 Bolton, E., and B. McCarthy, these PROCEEDINGS, 48, 1390 (1962).9 Gibson, I., and G. H. Beale, Genet. Res., 4, 42 (1963)."Reeve, E. C. R., and G. J. S. Ross, Genet. Res., 4, 158 (1963).1" Called to our attention by Barbara McManamy.12Jacob, F., and E. L. Wollman, Sexuality and Genetics of Bacteria (New York: Academic

Press, 1961), chap. 16, pp. 319-324.13Haruna, I., K. Nozu, Y. Ohtaka, and S. Spiegelman, these PROCEEDINGS, 50, 905 (1963);

Weissman, C., L. Simon, P. Borst, and S. Ochoa, Synthesis and Structure of Macromolecules, ColdSpring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 99; Baltimore, D., H. J.Eggers, R. M. Franklin, and I. Tamm, these PROCEEDINGS, 49, 843 (1963).

14 Wright, S., Am. Naturalist, 79, 289 (1945).

ENVIRONMENTAL CONTROL OF AMINO ACID SUBSTITUTIONSIN THE BIOSYNTHESIS OF THE ANTIBIOTIC

POLYPEPTIDE TYROCIDINE*

BY BERNARD MACH AND E. L. TATUM

LABORATORY OF BIOCHEMICAL GENETICS, THE ROCKEFELLER INSTITUTE

Read before the Academy April 27, 1964

Studies on the biosynthesis of tyrocidine, a bacterial decapeptide, have providedthe first example of the biosynthesis of a free polypeptide by mechanisms differentfrom those involved in the biosynthesis of proteins." 2 When several aspects ofprotein biosynthesis were studied in comparison with the biosynthesis of tyrocidine,it was demonstrated that (1) the enzymatic mechanisms involved in the incorpora-

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tion of amino acids, in the two types of syntheses, differ in their specific response tovarious amino acid analogues;' (2) inhibitors of protein synthesis such as chloram-phenicol, puromycin,l erythromycin, terramycin, and lincomycin' do not affect thesynthesis of the decapeptide; and (3) tyrocidine synthesis, in contrast to proteinsynthesis, does not depend on the continuous synthesis of RNA.4

It was suggested' that these small polypeptides, as well as other polypeptides ofthis size group, might be synthesized by an ordered stepwise addition of aminoacids directed by a series of specific enzymes, perhaps in a multienzyme complex.Some of these basic differences between the biosynthesis of protein and of smallpolypeptides have since been confirmed in the case of several other bacterial pep-tides, such as polymixin,5 gramicidin S,6 7 mycobacillin,8 and edeine.9

In the biosynthesis of proteins, the specificity of amino sequence is under directgenetic control, and the translation of a nucleotide sequence into a sequence of aminoacids apparently takes place with absolute specificity. Single amino acid substitu-tions observed in proteins such as hemoglobin, tryptophan synthetase, or bacterio-phage coat proteins are attributed to alterations in the nucleotide sequence ofstructural genes rather than to variations in environmental or metabolic conditions.Three forms of tyrocidine, A, B, and C (Table 1), have been described, which dif-

TABLE 1STRUCTURES OF TYROCIDINES A, B, C, AND D0, 11

A B C D

L-orn - L-leu -]D-he -Dhe -D-try -D-tryD- h II

Ival L-pro L-ro L-yro l1 Ioi T

L-tyr L-phe L-try L-try L-tryI ~ ~ ~ ~ ~ ~~~~~~~IIII

L-gluNH,-L-aspNH,-D-phe -D-phe -D-phe -D-try

fer from one another by single amino acid substitutions involving phenylalanineand tryptophan. 10 By analogy with protein synthesis, the amino acid substitutionsof tyrocidine might be under direct genetic control. Since a single culture of B.brevis produces three forms of tyrocidine (see Fig. 1), a direct genetic control wouldimply genetic heterogeneity of the culture with respect to the synthesis of tyroci-dines A, B, and C. Another less likely possibility would be the existence, in theDNA of each bacterial cell, of several templates differing from one another by singlenucleotide substitutions, and directing the simultaneous synthesis of the almostidentical tyrocidine polypeptides. Alternatively, in view of the basic differencesbetween the syntheses of protein and tyrocidine, it is also possible that these aminoacid substitutions are not the result of structural gene mutations, but depend uponenvironmental factors. This report establishes that the synthesis of the differentmolecular forms of tyrocidine is determined by the environmental concentration ofthe amino acids involved in these substitutions and ascribes this phenomenon toa low specificity of the enzyme systems involved in the incorporation of certainstructurally related amino acids.

Materials and Methods.-Bacterial strains, culture conditions, and the medium used were aspreviously described.' C14-L-Tyrosine, C14-L-leucine, and H3-L-tryptophan were purchasedfrom New England Nuclear Co. C'4-D-L-Ornithine was obtained from the California Corpora-tion for Biochemical Research. Tyrocidines A, B, and C, purified by countercurrent distribution,were a generous gift from Mr. M. Ruttenberg.

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The biosynthesis of tyrocidine was measured by the incorporation of radioactive amino acids byB. brevis cultures under well-defined conditions, followed by the isolation of tyrocidine, and thedetermination of specific activity.1

Tyrocidines A, B, and C were separated by gel filtration on a column of Sephadex G-25 (200 X1 cm) from which they are eluted on the basis of their tryptophan content.11 Elution was carriedout with 10% acetic acid at a rate of 10 ml/hr.

Bacterial clones from single spores: Appropriate dilutions of washed B. brevis spores were madein AGMM1 supplemented with 0.3% yeast extract. Glass slides were covered with mineral oil,and small drops of spore suspension were injected under the oil with a micropipette. The dropswere examined microscopically, and those containing one spore only were marked. The slideswere incubated at 370 overnight, and the bacterial clones obtained in the drops were used toinoculate 1 ml of medium.

B. brevis S-RNA and a crude activating enzyme fraction (105,000 X g supernatant) were ob-tained by published procedures.12

Formation of aminoacyl S-RNA: The incorporation mixture (1 ml) contained S-RNA (1 mg),crude enzyme fraction (1 mg protein) Tris-HCl buffer (pH 7.2) (100 pmoles), MgCl2 (10 Mmoles),ATP (10 /Amoles), and 0.4 usc of C14 amino acid (50 ,uc/Ismole). After incorporation at 360C for 8min, the samples were chilled in 0.25 N HC104, washed 3 times with 0.25 N HC104, and once eachwith ethanol-ether (1:3) and ether. After hydrolysis of the samples in 0.5 N HC104 at 900 for30 min, the radioactivity of the soluble portion was determined.

Results.-(1) Biosynthesis of tyrocidines A, B, and C: Following the incubationof a culture with radioactive amino acids and the isolation of tyrocidine, it is possi-ble to separate the different forms of the decapeptide by gel filtration and thus tostudy the biosynthesis of tyrocidines A, B, and C, respectively. RadioactiveL-leucine, D,L-ornithine, and L-tyrosine were used in these experiments with identi-cal results. It was first noted that a single culture of B. brevis synthesizes threedifferent forms of tyrocidine (Fig. 1). Furthermore, since the amino acid used

FIG. 1.-Synthesis of tyrocidinesA, B, and C. 20 ml of culture(ODwo m,:2.0) were incubated with

20 mnnules 24 hours C4-L-tyrosine (1 jsc/ml, 3 ,uc/p-c mole). After 20 min or 24 hr,

no 4 C 100 tyrocidine was isolated as described.g \ in Methods, dissolved in 1 ml or

x3 l 7.5 ~ ||510% acetic acid together with pureDo3. |-' '''150 unlabeled tyrocidines A, B, and C,2 A 5 and applied to a column of Sepha-i: .25~,~/U5 dex G-25. Elution was carried out

80 - 40 ; ; as described in Methods and the0 40 80 120 0 40 80 1L° 1co radioactivity was measured in ali-

Tube number quots of each tube. The dottedline (absorption at 280 mu) repre-sents tyrocidines A, B, and C,respectively.

as label is present in equimolar amount in the three forms of the peptide, the radio-activity of each tyrocidine peak is a direct measure of the relative amount of eachtyrocidine synthesized. After a 24-hr incubation with radioactive amino acids, theculture produces tyrocidines A, B, and C in a ratio of about 1:3:7 (Fig. 1). Thisratio is not due to preferential breakdown of tyrocidines A and B during the pro-longed incubation since the same result is obtained after very short time incorpora-tion (Fig. 1). By measuring the relative amounts of tyrocidines A, B, and C syn-thesized under a variety of conditions, it is possible to study the factors responsiblefor the single amino acid substitutions involved.

(2) Genetic homogeneity of the cultures: A possible explanation for the synthesis

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of three different forms of a polypeptide by a single culture could be the occurrenceof mutations during bacterial growth, resulting in a genetically heterogeneous cul-ture, with different cells synthesizing different tyrocidines. The relative amounts oftyrocidines A, B, and C produced by bacterial clones derived from single B. brevisspores were measured under conditions whichallowed only limited growth, and therefore a 61- Minimal mediumlower probability of mutations. The single- 5 Cspore cultures were grown in 1 ml of medium 4in the presence of radioactive amino acids 3 A(C14-L-tyrosine or C14-L-leucine, 10 Ac/0.4 2B,Omole/ml), and tyrocidine was isolated after A24 hr. In each of five separate single-sporeexperiments, each bacterial clone produced all AAthree forms of tyrocidine in exactly the same 12 - Minimalratio obtained from a 20-ml culture (see Fig. '° -+ L- Phe1). Furthermore, aliquots from 20- or 40-liter 8 -(O.5"umolecultures, in which a higher probability of muta- 6 /ml)tion would be expected, also produced tyroci- fO 4 Bdines A, B, and C in the same ratio. These ° 2 Cexperiments make it very unlikely that muta- I 0

tions occurring during the growth of a B. brevis ~ M mr r 1 1 * ^ 1 . ffi ~ ~~~~~~Minimal mediumculture account for the production of the three o 5 + L- Try

tyrocidines. They suggest a genetic homoge- (0.51.4mole/ml) Dneity of the cultures, with each cell synthesizingall different forms of tyrocidine. 3

(3) The effects of amino acids on the synthesis 2of tyrocidines A, B, and C: It was therefore of A B Cinterest to examine the effects of environmental 2040- 80 al1 140 16factors, such as the concentration of certain Tube numberamino acids, on the synthesis of tyrocidines A, A B C DB, and C. Although in the biosynthesis of PartilD D-Pile D-RPe D-Tryprotein an excess of one amino acid does not amino- L -PeLTry L -Try L-Trylead to its detectable incorporation in place of sequenceD D-Phe D-Try D-Tryanother, it is possible that such "errors" of in- __,_:____!_*_!corporation could take place in the synthesis FIG. 2.-Effect of L-amino acids onof tyrocidine. Since the differences between the biosynthesis of various forms of tyro-cidine. 20 ml of culture (OD600 mytyrocidines A, B, and C involve phe and try, 2.0) were incubated for 20 minthe effects of these two amino acids on the rela- with C'4-L-leucine (1 Mc/ml, 3 jUc/m-mole). L-phe or Itry were added,tive synthesis of the different tyrocidines were as indicated, 4 min before the radio-examined. As indicated (Fig. 2), the addition active amino acids. The different

medium r-. forms of tyrocidine were isolated andof L-phe to the culture in minimal medium re- separated as for Fig. 1.sulted in the almost exclusive synthesis oftyrocidine A (3 phe) at the expense of tyrocidines B and C. When L-try wasadded to the minimal medium, the phe-rich forms of tyrocidine were not synthe-sized and a new tyrocidine was produced instead, tyrocidine D, which containsthree try and no phe."1 This indicated the replacement of all three phe oftyrocidine A by try residues. If both L-phe and L-try were added, an inter-

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mediary ratio was obtained and the four forms of tyrocidine were synthesized.These experiments demonstrate that the availability, in the cellular environment,

of one or another of two structurally related amino acids determines which of thedifferent forms of tyrocidine is to be produced, and that this environmental factorcontrols the single amino acid substitutions involved.

(4) Low specificity of incorporation of structurally related amino acids: The ef-fects of phe and try indicate that the incorporation of one or another of two aminoacids at specific sites in a peptide chain depends on the relative concentration ofthese amino acids in the medium; this observation suggests that, in the biosynthe-sis of these small polypeptides, the enzymatic mechanisms involved in the activa-tion and incorporation of some structurally related amino acids cannot distinguishbetween them with absolute specificity. Several possible alternatives to this ex-planation should be considered.

If tyrocidine were produced by the biosynthetic mechanisms involved in proteinsynthesis, the observed effects of phe and try on the amino acid substitutions oftyrocidine would imply either a low specificity of activating enzymes or S-RNA's,an induction by the added amino acids of new activating enzymes or new S-RNA'swith different specificities, or a specific rate-limiting effect of certain amino acidsin a multitemplate situation. Although the incorporation of certain analogues ofamino acids into proteins has been demonstrated,"3 it is well known that thereis not competition for incorporation between the naturally occurring amino acidsof proteins. Large excesses of certain amino acids do not affect single amino acidsubstitutions involving these amino acids,'4 and the formation of aminoacyl-S-RNAin vitro is characterized by a rigorous specificity. 15 With B. brevis extracts obtainedfrom cultures identical to those used in the experiments on tyrocidine synthesis,the strict specificity of the acceptor ability of S-RNA toward phe and try wasconfirmed (Table 2). It was also shown, with the use of chloramphenicol and ac-

TABLE 2FORMATION OF AMINOACY-S-RNA BY EXTRACTS OF Bacillus brevis

Incorporation intoRadioactive amino acid Unlabeled amino acid aminoacyl-S-RNA(wimole)

C'4-L-phe 70.9 i±2.5C'4-L-phe L-try 71.0 4± 2.1Hs-L-try 43.9 ±00. 9H3-L-try L-phe 43.2 ± 1.2

The incubation mixture and the isolation of aminoacyl-S-RNA were as described in Methods. Un-labeled amino acids were added to a concentration ot 0.8 ;mole/ml. which represents 100 times theconcentration of the radioactive amino acids. Each experiment was performed in triplicate.

tinomycin, that neither protein nor RNA synthesis is involved in the observed ef-fects of phe and try on the synthesis of tyrocidines A, B, C, and D;16 these effectscan therefore not be attributed to an induction of either new activating enzymes ornew S-RNA's with modified specificities toward phe and try.

In protein synthesis the availability of specific amino acids is not a rate-limitingfactor in their incorporation. It was shown that the addition of a large excess ofeither phe or try (0.5 jAmole/ml every 2 hr) to a growing culture does not modifythe amino acid composition of total B. brevis proteins with respect to these twoamino acids.'7 Similar results have been obtained in E. coli with the same twoamino acids.'6 The effects of phe and try on the synthesis of the various forms of

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tyrocidine are therefore not compatible with the synthesis of these different poly-peptides, like proteins, on separate RNA templates, one for each different form oftyrocidine.

It appears that in the proposed series of enzymes that synthesize tyrocidine by astepwise addition of amino acids, three of these enzymes can incorporate either pheor try, depending on the availability of these two amino acids. An alternative ex-planation would be that each different form of tyrocidine (Table 1) is produced withgreat specificity by a distinct set of enzymes. If this were the case, the specificinhibition of phe incorporation into tyrocidine, by an amino acid analogue of phe,should decrease the synthesis of the phe-rich forms of tyrocidine, but should notaffect the synthesis of tyrocidine D, which contains no phe.It was observed, however, that threo-fl-phenylserine, a 7

competitive inhibitor of the incorporation of phe in tyro- 6Ccidine synthesis,18 inhibits the synthesis of all four forms of ° 4i _tyrocidine to the same extent, irrespective of their phe ca)

content, and that this analogue of phe inhibits the syn- O 4thesis of tyrocidine D which contains three try but no ,, 3 O

Ephe (Fig. 3). This observation indicates that the same E 2 menzymatic mechanisms, sensitive to threo-fl-phenylserine, iare responsible for the incorporation of either phe or tryat particular sites of the tyrocidine chain. It appears, °therefore, that the different tyrocidines are not produced FIG. 3.-Effect of phen-by different sets of highly specific enzymes, but rather by ysertine on the biosynthesisy ~~~~~oftyrocidine D. To in-one series of enzymes, some of which are capable of duce the synthesis of tyro-incorporating, at certain sites, either one of these two cidine D, L-try (0.5t Aomole/mi) was added to 10structurally related amino acids. ml of culture (see Fig.

(5) The effect of D-phe and D-try: It was seen that 2). After 3 min, threo-0-phenylserine (100 ug/-L-phe and L-try can affect amino acid substitutions involv- ml) was added, followeding the L as well as the D form of these amino acids (Fig. 2 min later by C'4-D-L-ingtheLaswellas the D form ~ornithine (0.8 s~c/ml, 32). Moreover, the addition of either D-phe or D-try ,uc/umole). Tyrocidineaffects the synthesis of the four different forms of tyrocidine synthesis was measured asin ref. 1.in a similar way (Fig. 4). Thus the incorporation of bothforms of phe can be modified by an excess of either of its two isomers, and thesame is true for try. Furthermore, L-phe-U-C'4 can be incorporated intotyrocidines B and C which contain only the D form of that amino acid. These re-sults are compatible with a racemization of these two amino acids and with a stereo-specific incorporation of one of the two isomers at specific sites in the polypeptide.

Discussion.-It has been demonstrated that in the biosynthesis of tyrocidine theamino acid substitutions observed at three sites in the decapeptide are determinedby the availability of the amino acids involved in these substitutions. Thus, anenvironmental factor, the level of an amino acid, can affect certain amino acidsequences of a polypeptide. This observation has several consequences.

(1) It was shown that the effects of phe and try on the amino acid substitu-tions of tyrocidine are not compatible with the mechanisms of amino acid activa-tion and incorporation involved in the biosynthesis of proteins. These experimentstherefore offer additional evidence for the involvement in tyrocidine synthesis ofa biosynthetic process distinct from that in protein synthesis.' It is of added in-

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terest that the replacement of phe by try, and vice versa, could not be attributed toany possible single-step nucleotide replacement in a structural gene according tothe published code letter assignments for the S-RNA's of these two amino acids. 19

(2) The competitive replacement of phe by try,51 Minimal medium and vice versa, at three different sites in the deca-4L- peptide tyrocidine can be attributed to a low spec-3k ificity of the enzymes involved in the recognition and2L B incorporation of amino acids at those three sites.IA This low specificity toward two structurally related

A amino acids can be compared with the incorporation6 Minimoli5 medium of hydroxyproline or sarcosine instead of proline in

04 O5Lmnie/n4) the formation of the peptide side chain of actino-n3 B mycin.20 A lack of absolute requirement for specific

-E 2 ~ A amino acids in the formation of a peptide bond is ofa c interest in comparison with the strict specificity ofthe sequential incorporation of amino acids into

imal medium proteins. In the formation of the peptide side chains5r +0-Try of S. aureus cell wall nucleotide2' and of actinomy-

cin,20 and presumably also in the synthesis of31- L) tyrocidine, the enzymatic mechanisms of peptide

bond formation in the stepwise addition of aminoacids are themselves responsible for the amino acid

20 460 80 00 120 140 160 specificity of the process. In the synthesis of proteins,FIG.Tubfetofbe Dhowever, the specificity of amino acid incorporation

acFIG.o4hEffecthessof -amino results from the participation of S-RNA moleculesforms of tyrocidine. Conditions and template RNA, and the enzymatic formationwere as desci ibed for Fig. 2. of peptide bonds need not be itself amino acid specific.

(3) The effects of the levels of certain amino acids on the synthesis of variousforms of tyrocidine could explain the simultaneous production of other small poly-peptides in different molecular forms. As in the case of bacitracin A and B (ileureplaced by val),22 the difference in the various forms of these peptides often con-sists in single amino acid substitutions involving structurally related amino acids;it is possible that, as in the case of tyrocidine synthesis, the production of thesedifferent molecular forms of the same peptide results from a low specificity of aminoacid incorporation reactions. It is of some interest that the production of both ly-sine-vasopressine and arginine-vasopressine has been recently observed in thesame animal species (peccary).23

(4) The reduced specificity toward structurally related amino acids observedin tyrocidine synthesis could be tested in studies on the mode of synthesis of othersmall polypeptides. Insulin, for instance, whose small chain consists of only twiceas many amino acids as tyrocidine, contains no try. It would be of interest todetermine if an excess of exogenous try would result in its substitution for phe in theinsulin molecule.

(5) The addition of try to cultures of B. brevis results in the synthesis of a newform of tyrocidine (tyrocidine D) which was found to have an antibiotic activityidentical to that of tyrocidines A, B, and C when tested against Neurospora crassa.24However, single amino acid modifications in the structure of certain polypeptide

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antibiotics or hormones can result in quantitative or qualitative changes in biologi-cal activity. The possibility of inducing the synthesis of new biologically activepolypeptides by modifications of the environmental conditions might therefore havepractical implications.

(6) Finally, reduced specificity in the incorporation of certain amino acidsmight apply to other biologically important polypeptides and would suggest thatthe exact amino acid sequence of these polypeptides is not under absolute geneticcontrol. Such a flexibility, and in particular the possibility of environmental fac-tors affecting the primary structure of a polypeptide chain, could have importantbiological implications. Modifications in physical or metabolic conditions of cells,which would result in a change in the level of certain amino acids, could determinethe synthesis of modified polypeptides, with distinct amino acid sequences. Such aflexible control of amino acid incorporation could provide an opportunity for en-vironmental factors to modify the structure and consequently to control the bio-logical activity of certain polypeptides. One could also postulate the existence ofother polypeptides of this type, characterized by the same flexible control of aminoacid sequence and capable, in addition, of binding rather specifically to certainmacromolecules: such polypeptides could play an important role in the regulation ofvarious cellular activities.The authors are very grateful to Dr. E. Reich for many stimulating discussions.* This investigation was supported in part by U.S. Public Health Service research grant CA-

03610, from the National Cancer Institute.1 Mach, B., E. Reich, and E. L. Tatum, these PROCEEDINGS, 50, 175 (1963).2 Mach, B., in Synthesis and Structure of Macromnobecules, Cold Spring Harbor Symposia on

Quantitative Biology, vol. 28 (1963), p. 263.3 Mach, B., and E. L. Tatum, manuscript in preparation.4Mach, B., and E. L. Tatum, Abstract V-I-219, Sixth International Congress of Biochemistry,

New York, 1964.Paulus, H., and E. Gray, J. Biol. Chem., 239, 865 (1964).

6 Eikhom, T. S., J. Jansen, S. Laland, and T. Refsvik, Biochim. Biophys. Acta, 76, 465 (1963).7Ibid., 80, 648 (1964).8 Barnerjee, A. B. and S. K. Bose, J. Bacteriol., 87, 1397 (1964).9 Borowska, S., and E. L. Tatum, to be published.

10 King, T. P., and L. C. Craig, J. Am. Chem. Soc., 77, 6627 (1955).11 Ruttenberg, M., and B. Mach, manuscript in preparation.12 Nirenberg, M., and J. H. Matthaei, these PROCEEDINGS, 47, 1588 (1961).13 Cohen, G. N., and F. Gros, Ann. Rev. Biochem., 29, 515 (1960).14 Brody, S., and C. Yanofsky, personal communication.15 Loftfield, R. B., Federation Proc., 22, 644 (1963).16 The experiment described in Fig. 2 was repeated after addition of either chloramphenicol

(50 ,sg/ml) or actinomycin D (10 Ag/ml) 3 min before L-phe or b-try. Under these circumstances,the synthesis of protein and RNA, respectively, is inhibited;3 the biosynthesis of the various formsof tyrocidine, however, was unaffected and the results were the same as those reported in Fig. 2.

17 Mach, B., and M. Ruttenberg, unpublished results.18 The effect of phenylserine on tyrocidine synthesis is reversed by phenylalanine. This re-

versal is not due to competition for uptake since tyrosine does not reverse the phenylserine in-hibition.

19 Nirenberg, M. W., 0. W. Jones, P. Leder, B. F. C. Clark, W. S. Sly, and S. Pestka, in Synthesisznd Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28(1963), p. 263.

20 Katz, E., Ann. N. Y. Acad. Sci., 89, 304 (1960).21 Strominger, J. L., Federation Proc., 21, 134 (1962).

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22 Craig, L. G., W. Koenigsberg, and R. J. Hill, Amino Acids and Peptides with AntimetabolicActivity, CIBA Foundation Symposium (1958), p. 226.

23 Du Vigneaud, V., personal communication.24 Mach, B., C. Slayman, and E. L. Tatum, manuscript in preparation.

TRYPSINOGEN AND CHYMOTRYPSINOGEN ASHOMOLOGOUS PROTEINS

BY KENNETH A. WALSH AND HANS NEURATH

UNIVERSITY OF WASHINGTON, SEATFLE

Communicated August 27, 1964

The striking similarity of many properties of trypsin and chymotrypsin has beenwell known for many years.1-5 Both enzymes originate in the acinar cells of pan-creatic tissue6 at identical rates7 as their inactive precursors trypsinogen and chymo-trypsinogen A, and both are activated by the cleavage (by trypsin) of the peptidebond contributed by the basic residue which is closest to the amino terminus of thezymogen.8, 9 Both enzymes catalyze the hydrolysis of peptide, amide, and esterbonds, and both are characterized by a high degree of selectivity toward the aminoacids which donate carboxyl groups to these bonds.10-'2 In both cases, the enzymesappear to become acylated through these carboxyl groups as an intermediate stepin the catalytic mechanism.."-"5 Both enzymes are inhibited by certain organicfluorophosphates such as DFP, 16 and the site of reaction of these inhibitors has beenidentified as a unique serine residue in the same tetrapeptide sequence.17' 18 Thesame serine residue becomes acylated as an intermediate in the catalytic mecha-nism."9 In addition to serine, a histidine residue has long been implicated as acomponent of the active site of both chymotrypsin and trypsin.20 Indirect supportingevidence, derived from the pH dependence of the acylation and deacylation steps,and from the effects of photooxidation on enzyme activation of chymotrypsin,2' hasbeen recently strengthened and confirmed by the findings of Shaw and co-workers22-24 that bifunctional reagents, resembling in structure specific substrates,inactivate chymotrypsin and trypsin, respectively, by forming covalent derivativesof a single histidine residue in each enzyme.

Chymotrypsinogen and trypsinogen resemble each other in certain chemicalproperties.1 These include molecular weights (approximately 25,000 and 24,000,respectively) and isoelectric points (approximately 9.3). A comparison of theamino acid compositions of the two zymogens25-27 in Table 1 reveals that the similarityextends to this level also. The compositional similarity is made even more strikingif certain residues of similar chemical character are grouped together, such as thetotal acidic, total basic, total aromatic, total branched hydrophobic, and totalhydroxy-amino acid residues. Such comparative data are given at the bottomof Table 1.The only really striking difference between the two enzymes lies in their sub-

strate specificity." Whereas trypsin acts almost exclusively on peptide bondsinvolving the carboxyl groups of lysine or arginine residues, chymotrypsin has nosignificant action upon these bonds, but is most active toward linkages involving the

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