specificity." Whereas trypsin acts almost exclusively on peptide bonds properties.1 These include molecular weights (approximately 25,000 and 24,000,

Transcription

1 884 BIOCHEMISTRY: WALSH AND NEURATH PROC. N. A. S. 22 Craig, L. G., W. Koenigsberg, and R. J. Hill, Amino Acids and Peptides with Antimetabolic Activity, CIBA Foundation Symposium (1958), p Du Vigneaud, V., personal communication. 24 Mach, B., C. Slayman, and E. L. Tatum, manuscript in preparation. TRYPSINOGEN AND CHYMOTRYPSINOGEN AS HOMOLOGOUS 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 been well known for many years.1-5 Both enzymes originate in the acinar cells of pancreatic tissue6 at identical rates7 as their inactive precursors trypsinogen and chymotrypsinogen A, and both are activated by the cleavage (by trypsin) of the peptide bond contributed by the basic residue which is closest to the amino terminus of the zymogen.8, 9 Both enzymes catalyze the hydrolysis of peptide, amide, and ester bonds, and both are characterized by a high degree of selectivity toward the amino acids which donate carboxyl groups to these bonds.10-'2 In both cases, the enzymes appear to become acylated through these carboxyl groups as an intermediate step in the catalytic mechanism.."-"5 Both enzymes are inhibited by certain organic fluorophosphates such as DFP, 16 and the site of reaction of these inhibitors has been identified as a unique serine residue in the same tetrapeptide sequence.17' 18 The same serine residue becomes acylated as an intermediate in the catalytic mechanism."9 In addition to serine, a histidine residue has long been implicated as a component of the active site of both chymotrypsin and trypsin.20 Indirect supporting evidence, derived from the ph dependence of the acylation and deacylation steps, and from the effects of photooxidation on enzyme activation of chymotrypsin,2' has been recently strengthened and confirmed by the findings of Shaw and coworkers22-24 that bifunctional reagents, resembling in structure specific substrates, inactivate chymotrypsin and trypsin, respectively, by forming covalent derivatives of a single histidine residue in each enzyme. Chymotrypsinogen and trypsinogen resemble each other in certain chemical properties.1 These include molecular weights (approximately 25,000 and 24,000, respectively) and isoelectric points (approximately 9.3). A comparison of the amino acid compositions of the two zymogens25-27 in Table 1 reveals that the similarity extends to this level also. The compositional similarity is made even more striking if certain residues of similar chemical character are grouped together, such as the total acidic, total basic, total aromatic, total branched hydrophobic, and total hydroxy-amino acid residues. Such comparative data are given at the bottom of Table 1. The only really striking difference between the two enzymes lies in their substrate specificity." Whereas trypsin acts almost exclusively on peptide bonds involving the carboxyl groups of lysine or arginine residues, chymotrypsin has no significant action upon these bonds, but is most active toward linkages involving the

2 VOL. 52, 1964 BIOCHEMISTRY: WALSH AND NEURATH 885 TABLE 1 AMINO ACID COMPOSITIONS OF TRYPSINOGEN26 AND CHYMOTRYPSINOGEN25 Trypsinogen Chymotrypsinogen Asparagine Glutamine Aspartic acid 10 9 Threonine t Serine Glutamic acid 3 5 Proline 9 9 Glycine Alanine Cystine/ Valine Methionine 2 2 Isoleucine Leucine Tyrosine 10 4 Phenylalanine 3 6 Tryptophan 4 8 Lysine Histidine 3 2 Arginine 2 4 Total acidic residues Lysine and arginine Total amides Total aromatic residues Serine + threonine Sum of isoleucine, leucine, and valine carboxyl groups of aromatic amino acids. Although trypsin also catalyzes the cleavage of certain aromatic substrates (characteristic of chymotrypsin), it is less effective than chymotrypsin in this regard, and far less effective than toward its own basic substrates. Several lines of evidence indicate that this property is intrinsic in trypsin itself, and not the result of a chymotryptic contamination.28 The recent finding of Mares-Guia and Shaw29 that a binding site for aromatic compounds exists at the active center of trypsin indicates that the striking difference in specificity between the two enzymes may turn out to be a quantitative phenomenon, rather than a qualitative one, and draws attention again to the underlying similarity between the two enzymes. The fundamental similarities in biological, mechanistic, and compositional character between the two molecules have led to the logical prediction of an extensive similarity in primary structure,5 including in particular the regions of structure known to have functional importance. However, as the primary structure of the two molecules was evolved,30 no analogous amino acid sequences were found that were larger than the tetrapeptide sequence originally observed to surround the serines of the active centers. More recently,3"-33 a nearly identical nonadecapeptide sequence was observed in chymotrypsin and trypsin containing two histidine residues in sufficiently close proximity to each other to suggest that not one but both of them are functional components of the active center in both enzymes.26 The recent completion of the structure of chymotrypsinogen A25 and a tentative structure for trypsinogen27 31 permit a detailed comparison of the molecular structures of the two enzymes, and a search for evidence of homology. This comparison is made in Table 2 by outlining the linear structures of the two zymogens in such a fashion as to make in each the amino terminal isoleucine residue, formed

3 886 BIOCHEMISTRY: WALSH AND NEURATH PROC. N. A. S. Chymotrypsinogen Trypsinogen TABLE 2 THE STRUCTURAL SIMILARITY OF TRYPSINOGEN AND CHYMOTRYPSINOGEN cys-gly-val-pro-ala-ile-gln-pro-val-leu-ser-gly-leu-ser-arg-ile-val-glyval-asp-asp-asp-asp-lys-ile-val-gly asp-glu-glu-ala-val-pro-gly-ser-trp-pro-trp-gln-val-ser-leu-gln-asp-lys-thr-gly-phe-his-phegly-tyr-thr-cys-gly-ala-asn-thr-val-pro-tyr-gln-val-ser-leu-asn- -ser-gly-tyr-his-phe_ CYS-GLY-GLY-SER-LEU-ILE-ASN-glu-asn-TRP-VAL-VAL-thr-AIA-ALA-HIS-CYS-gly-val-thr-thr-ser-asp- CYS-GLY-GLY-SER-LEU-ILE-ASN-ser-gln-TRP-VAL-VAL-ser-AIA-ALA-HIS-CYS-tyr-lys-ser-gly-ile-gln VAL-val-val-ala-gly-glu-phe-asp-gln-gly-ser-ser-ser-glu-lys-ile-gln-lys-leu-lys-ile-ala-lys- VAL-arg-leu-gly-glu-asp-asn-ile-asn-val-val-glu-gly-asp-glu-gln-phe-ile-ser-ala-ser-lys-ser , val-phe-lys-asn-ser-lys-tyr-asn-ser-leu-thr-ile-asn-asn-asn-ile-thr-leu-leu-lys-leu-ser-thrile-val-his-pro-ser- -TYR-ASN(pro,leuthr,asn)ASN-ASN-asp-ILE-met-LEL-ile-LYS-LEU-lys-ser go AIA-ALA-SER-phe-ser-gln-thr-VAL-ser-ala-val-cys-LEU-PRO-ser-ala-ser-asp-asp-phe-ala-AIA-GLY- AIA-ALA-SER-leu-asn-ser-arg-VAL-ala-ser-ile-ser-LEU-PRO-thr-ser-cys- -41a-ser-AIA-GLY o THR-thr-CYS-val-thr-thr-GLY-TRP-GLY-leu-THR-arg-tyr-thr-asn-ala-asn-thr-PRO-ASP-arg-LEU-gln- THR-gln-CYS-leu-ile-ser-GLY-TRP-GLY-asn-THR-lys-ser-ser-gly-thr-ser-tyr-PRO-ASP-val-LEU-lys gln-ala-ser-leu-pro-leu-leu-ser-asn-thr-asn-cys-lys-lys-tyr-trp-gly-thr-lys-ile-lys-asp-ala- cys-leu-lys-ala-pro-ile-leu-ser-asp-ser-ser-cys-lys-ser-ala-tyr-pro-gly-gln-ile-thr-ser-asn MET-ile-CYS-AIA-GLY-ala-ser-gly-val-ser- -SER-CYS-met-GLY-ASP-SER-GLY-GLY-PRO-leu-VAIL. MET-phe-CYS-ALA-GLY-tyr-leu-glu-gly-gly-lys-asn-SER-CYS-gln-GLY-ASP-SER-GLY-GLY-PRO-val-VAL lj CYS-lys-lys-asn-gly-ala-trp-thr-leu-val-GLY-ILE-VAL-ser-SER-TRP-GLY-SER-ser-thr-cys-ser-thr-,CYS-ser-gly-lys-leu-gln- -GLY-ILE-VAL- -SER-TRP-GLY-SER-gly-cys-ala-gln-lys I ser-thr-pro-gly-val-tyr-ala-arg-val-thr-ala-leu-val-asn-trp-val-gln-gln-thr-leu-aia-ala-asn asn-lys-pro-gly-val-tyr-thr-lys-val-cys-asn-tyr-val-ser-trp-ile-lys-gln-thr-ile-ala-ser-asn The sequence of chymotrypsinogen is taken from Hartley25 and the tentative structure of trypsinogen27 from Walsh, Sampath Kumar, and Kauffman.U The positions of the disulfides are not indicated. The sequence in trypsinogen has not yet been established. Gaps in the sequence have been arbitrarily inserted to draw attention to the resulting areas of homology. Amino acid residues that are homologous in the two proteins are shown in capital letters. upon activation, a common starting point. The rationale for this basis of comparison is derived from the analogies in the activation of the two zymogens, which include, among others, the formation of an identical isoleucyl-valyl-glycyl N- terminal sequence in the newly formed enzymes.34' 26 The mutual alignment of the two linear structures shown in Table 2 in addition makes allowance for occasional deletions following a precedent set by the hemoglobin-myoglobin com-

4 VOL. 52, 1964 BIOCHEMISTRY: WALSH AND NEURATH 887 parisons.35 The data of Table 2 indicate that approximately 40 per cent of the amino acid residues occur in identical positions in the two chains. Identical triand even dipeptide sequences which previously have seemed statistically unimportant36 now indicate a striking homology because they are located in analogous positions throughout the two molecules. This type of homology could not be detected until the complete structure of both proteins was known. Allowing for minor amino acid replacements, the longest continuous sequence common to both proteins is the 19-amino-acid peptide containing two histidines, and extending from histidine -29 in trypsinogen (40 in chymotrypsinogen A) to half-cystine 47 in trypsinogen (58 in chymotrypsinogen). The other common area of functional importance extends, with minor modifications, from serine 178 in trypsinogen (190 in chymotrypsinogen A) to half-cystine 189 in trypsinogen (201 in chymotrypsinogen A) and contains in the middle the reactive serine as part of the previously known, common tetrapeptide sequence Gly-Asp-Ser-Gly. The homology in this region is more extensive than previously believed, because of a previous error in the sequence of the active center peptide37 isolated from trypsin. Close examination of the apparent homology of the two proteins indicates some gradation in the frequency of some amino acid replacements, which may well reflect the importance of these residues to the conformation of the proteins. Thus, it may be of structural significance that 6 of the 7 proline residues in the region of chymotrypsin subject to comparison are in identical positions in trypsin, and all 4 tryptophan residues in trypsinogen occur in identical positions in chymotrypsinogen. On the other hand, 50 of the 62 charged residues of both molecules (lysine, arginine, aspartic, and glutamic acid residues) do not occur in homologous positionsan observation which would be consistent with their location on the outer surface of the molecules. Seven or eight of the 10 half-cystines in chymotrypsinogen are found in homologous loci in trypsinogen. The two nonhomologous half-cystines in chymotrypsinogen (nos. 1,122) have been shown to be joined by a disulfide bond.38 Four of the eight homologous half-cystines of chymotrypsinogen have been tentatively shown to be identically paired by homologous disulfide bonds in the two zymogens3l (nos. 42, 58, and nos. 168, 182 in chymotrypsinogen). However, the further extent of this homology cannot be established until the positions of the remaining disulfide bridges in trypsinogen have been completely elucidated. 27 The degree of conservation of character observed with the two enzymes is not as great as that found in either the hemoglobins35 or the cytochromes,39 but the more extensive structural differences between chymotrypsinogen and trypsinogen are accompanied by a clear change in biological specificity, whereas the specific biological function of the hemoproteins is maintained, in spite of structural deviations. While the actual homology between trypsinogen and chymotrypsinogen is approximately 40 per cent, a homology of character can be seen to extend further if one equates certain amino acid residues having analogous chemical properties. For example, if one equates serine to threonine, and leucine to isoleucine to valine, the homology is extended to 50 per cent of the molecule. If one further extends this principle by equating the aromatic amino acids with each other, the lysine and arginine residues with each other, and the asparagine and glutamine residues with each other, the homology extends to 56 per cent of the residues. It would be difficult to ignore the chemical differences between these residues except by in-

5 888 BIOCHEMISTRY: WALSH AND NEURATH Pioc. N. A. S. dicating that a gross change in chemical nature (and conformational consequences) would not be involved in any of these hypothetical transformations. The high degree of conservation inherent in this homology must reflect either areas of the molecule which have evolved spontaneously and independently by virtue of their necessity for the induction of proteolytic activity by whatever mechanism, or areas which have persisted in the course of evolution of the two proteins from a common ancestral precursor because they are necessary for function. It is difficult to accept the former explanation, because it would necessitate that 40 per cent of each molecule be directly or indirectly involved in the actual function of the enzyme. On the other hand, the latter explanation requires only that certain areas which are of functional importance should not change, but dictates nothing about the degree of change or lack thereof in the remainder of the molecule. If the theory is valid that these two enzymes have evolved from a common precursor, there has been in the course of evolution not only a change of 60 per cent of the molecule, and a consequential marked change in specificity, but also at the same time an equally marked preservation of both the basic physicochemical character of the molecule, and the fundamental catalytic mechanism. From the viewpoint of enzymatic specificity, the differences in covalent structure are more important than their similarities. Somewhere along the sequence of the two enzymes there must be an array of amino acid residues specific for each enzyme, and distributed in such a manner as to give rise in the tertiary structure to the configuration characteristic for tryptic and chymotryptic specificity, respectively. Unfortunately, no method of labeling has yet been found which would identify in the degraded enzyme the residues responsible for the specific substrate binding, and hence lend itself to direct solution in the manner of the present analysis. It can be approached, however, by comparison of the covalent structure of enzymes having similar specificity and occurring in various species of animals.32 Some homology is clearly indicated between bovine and porcine trypsinogen,40 and between bovine chymotrypsinogens A and B.34 It seems a reasonable prediction that the similarities of composition, end-groups, and activation between bovine and porcine trypsin would extend to the level of the structural homology. The recent findings of proteolytic activity with parallel specificities in the pancreas of lower animals (dogfish)4' suggest the feasibility of exploring the extent of homology in a particular enzyme as it evolved, concurrent with the appearance of individual species of animals. At this stage, it seems probable that one can anticipate a parallel between paleontological evidence for the order of evolution of biological structure, that is, of species, and concurrent evolution of chemical structure, that is, the specificity and mechanism of enzymes. Perhaps, in fact, one must anticipate a closer relationship between many proteins now thought to be completely unrelated. ' Green, N. M., and H. Neurath, in The Proteins, ed. H. Neurath and K. Bailey (New York: Academic Press, 1954), vol. 2B, p Desnuelle, P., in The Enzymes, ed. P. D. Boyer, H. Lardy, and K. Myrback (New York: Academic Press, 1960), vol Neurath, H., G. H. Dixon, and J.-F. Pechere, Proc. Intern. Congr. Biochem. 4th, 1960, 63 (vol. 8). 4 Hartley, B. S., Ann. Rev. Biochem., 29, 45 (1960). 6 Sorm, F., and B. Keil, Advan. Protein Chem., 17, 167 (1962). 6 Siekevitz, P., and G. Palade, J. Biophys. Biochem. Cytol., 7, 619, 631 (1960).

6 VOL. 52, 1964 BIOCHEMISTRY: WALSH AND NEURATH 889 7Keller, P. J., E. Cohen, and H. Neurath, J. Biol. Chem., 234, 311 (1959). 8 Neurath, H., Advan. Protein Chem., 12, 320 (1957). 9 Neurath, H., and G. H. Dixon, Federation Proc., 16, 791 (1957). 10 Bergmann, M., and J. S. Fruton, Advan. Enzymol., 1, 63 (1941). 11 Neurath, H., and G. W. Schwert, Chem. Rev., 46, 70 (1950). 12 Hein, G., and C. Niemann, these PROCEEDINGS, 47, 1341 (1961). 13 Hartley, B. S., and B. A. Kilby, Biochem. J., 56, 288 (1954). 14 Gutfreund, H., and J. M. Sturtevant, these PROCEEDINGS, 42, 719 (1956). 16 Bender, M. L., and E. T. Kaiser, J. Am. Chem. Soc., 84, 2557 (1962). 16 Jansen, E. F., and A. K. Balls, J. Biol. Chem., 194, 721 (1952). 17 Schaffer, N. K., C. S. May, Jr., and W. H. Summerson, J. Biol. Chem., 202, 67 (1953). 1 Dixon, G. H., D. L. Kauffman, and H. Neurath, J. Biol. Chem., 233, 1373 (1958). 19 Oosterbaan, R. A., and M. E. Van Adrichem, Biochim. Biophys. Acta, 27, 423 (1958). 20 Dixon, G. H., H. Neurath, and J.-F. Pech~re, Ann. Rev. Biochem., 27, 489 (1958). 21 Koshland, D. E., Jr., D. H. Strumeyer, and W. J. Ray, Jr., in Enzyme Models and Enzyme Structure, Brookhaven Symposia in Biology, No. 15 (1962), p Schoellmann, G., and E. Shaw, Biochemistry, 2, 252 (1963). 23 Mares-Guia, M., and E. Shaw, Federation Proc., 22, 528 (1963). 24 Ong, E. B., E. Shaw, and G. Schoellmann, J. Am. Chem. Soc., 86, 1271 (1964). 25 Hartley, B. S., Nature, 201, 1284 (1964). 26 Walsh, K. A., D. L. Kauffman, K. S. V. Sampath Kumar, and H. Neurath, these PROCEED- INGS, 51, 301 (1964). A series of papers giving the complete proof of structure of bovine trypsinogen is in preparation, to be published in Biochemistry. 28 Inagami, T., and J. M. Sturtevant, J. Biol. Chem., 235, 1019 (1960). 29 Mares-Guia, M., and E. Shaw, Abstracts, 6th International Congress of Biochemistry, 1964, vol. 4, p Walsh, K. A., D. L. Kauffman, and H. Neurath, Federation Proc., 20, 385 (1961). '7 Walsh, K. A., K. S. V. Sampath Kumar, and D. L. Kauffman, Abstracts, 6th International Congress of Biochemistry, 1964, vol. 2, p Neurath, H., Abstracts, 6th International Congress of Biochemistry, 1964, vol. 4, p Hartley, B. S., Abstracts, 6th International Congress of Biochemistry, 1964, vol. 4, p Desnuelle, P., and M. Rovery, Advan. Protein Chem., 16, 139 (1961). 3 Braunitzer, G., R. Gehring-Muller, N. Hilschmann, K. Hilse, G. Hobom, V. Rudloff, and B. Wittmann-Liebold, Z. Physiol. Chemie, 325, 283 (1961). 36 Williams, J., J. B. Clegg, and M. 0. Mutch, J. Mol. Biol., 3, 532 (1963). 37 Dixon, G. H., D. L. Kauffman, and H. Neurath, J. Biol. Chem., 233, 1373 (1958). 38 Brown, J. R., and B. S. Hartley, Biochem. J., 89, 59P (1963). 39 Margoliash, E., these PROCEEDINGS, 50, 672 (1963). 40 Charles, M., M. Rovery, A. Guidoni, and P. Desnuelle, Biochim. Biophys. Acta, 69, 115 (1963). 41 Prahl, J. W., and H. Neurath, Abstracts, 142nd National Meeting of the American Chemical Society, 1962.