Relationships to Carboxypeptidase a (Amino Acid Sequence/Active Site/Proteolytic Cleavage) GERALD R

Proc. Nat. Acad. Sci. USA Vol. 68, No. 6, pp. 1226-1230, June 1971

New Forms of Bovine Carboxypeptidase B and Their Homologous Relationships to Carboxypeptidase A (amino acid sequence/active site/proteolytic cleavage) GERALD R. REECK, KENNETH A. WALSH, MARK A. HERMODSON, AND HANS NEURATH Department of Biochemistry, Uriiversity of Washington, Seattle, Wash. 98105 Communicated Janutary 29, 19711

ABSTRACT Two new forms of carboxypeptidase B MATERIALS AND METHODS have been isolated from spontaneously activated bovine bovine pan- pancreatic juice. The fully active enzymes contain an For the isolation of bovine carboxypeptidase B, internal split at residues 92-93 and 95-96, respectively. creatic juice was collected (with the generous cooperation of Sequenator analysis of the amino terminal segments of Dr. R. Sande) at the School of Veterinary Science, Washing- the two chains of the enzyme has extended the sequence ton State University, Pullman, Wash., by the general pro- information by 51 amino acid residues. Comparison of 125 residues strengthens the hypothesis that carboxypeptidases A and B are homologous both in amino acid sequence and in three-dimensional conformation and implicates Asp-255 as the anionic site of substrate binding of the B enzyme. Bovine carboxypeptidase isolated from partially purified B, 0 procarboxypeptidase B (1), is composed of a single polypep- CN tide chain containing approximately 300 amino acid residues. The enzyme resembles carboxypeptidase A in molecular weight, in certain features of its amino acid composition, in metal content, and in its mechanism of action (2). The two enzymes differ in substrate specificity. We have recently pro- posed that bovine carboxypeptidases B and A are homologous both in amino acid sequence and in three-dimensional config- uration (3). In order to extend the experimental proof of homology, we undertook additional structural investigations of B. Unforeseen difficulties in un- carboxypeptidase securing a.I autolyzed bovine pancreatic juice directed our efforts toward I' the isolation of the enzyme from spontaneously activated r ' pancreatic secretions. The final products differed from the previously known enzyme in having been converted by limited proteolytic cleavage into new, fully active forms whose general characteristics and structural relationships to carboxypep- 40 60 80 tidase A are described in this report. Fraction Number

0.6- C FIG. 1. Purification of carboxypeptidase B. (A) Chrom- atography on e-aminocaproyl-D-tryptophan-Sepharose 4B of o the exopeptidase fraction from the soybean trypsin inhibitor- Ad 0.3- Sepharose 4B column. The initial eluent was 0.02 M Tris (pH 0.1 of were 7.5)-0.05 M NaCl. The higher concentrations NaCl also t0.05 [NoC]j in solutions of 0.02 M Tris, pH 7.5. Fraction volume was 20 ml. Y (B) Assays of chromatographic fractions of carboxypeptidases I-) B and A using carbobenzoxyglycyl-L-phenylalanine and benzoyl- -ZN glycyl-i.arginine, respectively. (C) Chromatography on DE-52 1. cellulose of the carboxypeptidase B-containing fractions from Cz the preceding chromatography. The initial eluent was 0.02 M 1. ";;k Tris, pH 8.0. A linear gradient from 0 to 0.1 M NaCl was applied. I'I.i The column size was 2.5 X 40 cm and the flow rate was 200 ml/ 20 40 60 80 100 hr. Fraction volume was 20 ml. (D) Carboxypeptidase B assays 01) of the chromatographic fractions of C. Fraction Number 1226 Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Structure of Carboxypeptidases A and B 1227 f

ffiSlRK

FIG. 2. Left, polyacrylamide disc ....S gel electrophoresis (8) of carboxypepti- AS _w ::_mI dase B from active bovine pancreatic _ _: juice. From left to right: product of ...... : . .7:So 41-p- Fig. 1A; Fraction I of Fig. iC; Frac- I tion II of Fig. 1C; Fractions I and II of : .;

Fig. iC mixed before electrophoresis...... ,. id Right, polyacrylamide disc gel electrophoresis (9) in the presence of sodium > I'.. dodecyl sulfate and fl-mercaptoethanol. _-a_ _:_ ghi: From left to right: Fraction I of Fig. it': iC; Fraction II of Fig. IC; native :, carboxypeptidase B (1); Fraction I of Fig. IC mixed with native carboxy- :. peptidase B before electrophoresis.

* a

U t `:1- -,.- ',. i;A

j -saft.-.. cedure of Keller et al. (4). Despite antiseptic precautions and 35 cm column of insoltubilized soybean trypsin inhibitor, which rapid freezing of the collected juice, the zymogens were fully had been prepared by covalent coupling of inhibitor to Sephar-

activated on arrival at the University of Washington. ose 4B activated with cyanogen bromide (5). The exopepti- As a first step the endopeptidases were removed by chromatography of 1600 ml of untreated pancreatic juice on a 5 X TABLE 1. - nalysis of homologous loci* in bovine A. "Light chain" of Fraction II carboxypeptidases A and B 10 THR-THR-GLY-HIS-SER-TYR-GLU-LYS-TYR-ASN- Location of residue in CPAt 20 Surface Interior ASN-TRP-GLU-THR-ILE-GLU-ALA-TRP-THR-GLU 30 Re- Re- GLN -VAL -ALA-SE R-GL U-ASN-PR0-ASP-LEU- I LE Nature of residue Identical placed Identical placed in in in in In CPA In CPB CPB CPB CPB CPB B. "Heavy chain" of Fraction II Polar Polar 13 24 6 1 10 Hydro- Hydro- 16 9 12 20 GLU-ILE-HIS-MET-THR-GLU-PHE-LEU-ASP-LYS- phobic: phobic 19 Polar Hydro- 7 5§ LEU-ASP-PHE-TYR-VAL-LEU-PRO-VAL-VAL phobic Hydro- Polar 9 3 phobic 49 18 29 C. "Heavy chain" of Fraction I Total 29

MET-THR-GLU-PHE-LEU-ASP-LYS- * Homologous loci as indicated in Fig. 4 (except residues 277- 279 and 308). LEU-ASP-PHE-TYR-VAL-LEU-PRO-VAL-VAL-ASN- t Location of residues deduced from the three-dimensional structure derived by Lipscomb et al. (18). ILE-ASP-GLY-TYR-ILE-TYR $ Hydrophobic residues are Pro, Gly, Ala, Cys, Val, Ile, Leu, Met, Tyr, Phe, and Trp. The rest are considered polar. FIG. 3. Amino-terminal sequences of separated fragments of § The five interior residues that are polar in carboxypeptidase spontaneously activated bovine carboxypeptidase B. The frag- A and hydrophobic in carboxypeptidase B are: Ser7o- Ala; ments were separated on Sephadex G-75 in 4 M urea-10% acetic Gln76 -Pro; Thr78 - Phe; Thr87- Val; Thrill - Val. Other than acid. The sequence of the light chain of Fraction I was identical the change at residue 78, the volume changes are small and the with that of the light chain of Fraction II. When either Fraction hydrophobic nature of the residues in carboxypeptidase B would I or Fraction II (from Fig. 1C) was analyzed in the sequenator be expected to fit into a hydrophobic interior. prior to separation of the fragments, phenylthiohydantoin- ¶ The three interior residues that are hydrophobic in carboxy- amino acids were released two at a time, corresponding to simul- peptidase A and polar in carboxypeptidase B are: Ile255- Asp; taneous degradations of one light and one heavy chain. Val8o - Gln; Met22- Thr. Downloaded by guest on September 26, 2021 1228 Biochemistry: Reeck et al. Proc. Nat. Acad. Sci. USA 68 (1971)

15 FIG. 4. Comparison of the partial structure of carboxypepti- CPB THR-THR-GLY-H IS-SER-TYR-GLU-LYS-TYR-ASN-ASN-TRP- dase B with portions of the complete structure of carboxypepti- CPA ALA-ARG-SER-THR-ASN-THR-PHE-ASN-TYR-ALA-THR-TYR-HIS-THR-LEU- dase Aa (12). Sequences in carboxypeptidase B corresponding to 30 specific sequences in carboxypeptidase A are derived from the GLU-THR- I LE-GLU-ALA-TRP-THR-GLU-GLN-VAL-ALA-SER-GLU-ASN-PRO- following sources and placed in alignment by considerations of ASP-GLU- ILE-TYR-ASP-PHE-MET-ASP-LEU-LEU-VAL-ALA-GLU-HIS-PRO- structural homology (the numbers refer to specific residue num- 33 bers in bovine carboxypeptidase A): ASP-LEU-ILE- - - - Residues 4-33: Derived from sequence analysis of the "light" GLN-LEU-VAL- chain of Fraction II (Fig. 3). The alignment was established by an analysis for homology by a computer method (G. R. Reeck, 75 - MET-ASP-CYS-GLY-PHE-HIS-ALA-ARG-GLU-TRP-ILE-SER- unpublished). The amide placement at positions 28 and 31 of ALA-ILE-TRP-ILE-ASP-LEU-GLY-ILE-HIS-SER-ARG-GLU-TRP-ILE-THR- carboxypeptidase A has been corrected (P. H. Petra, M. A. Hermodson, K. A. Walsh, and H. Neurath, in preparation). 90 PRO-ALA-PHE-CYS -GLN-TRP-PHE-VAL-ARG-GLU-ALA-VAL-ARG-THR-TYR- Residues 64-96: A peptide "CN-2" with the sequence of residues 65-96 was isolated from carboxypeptidase B after cleavage GLN -ALA-THR-GLY-VAL-TRP-PHE-ALA-LYS -LYS-PHE-THR-GLU-ASN-TYR- with cyanogen bromide (11); Met-64 was placed by virtue of the 105 specificity of cleavage by cyanogen bromide; the alignment with GLY-ARG-GLU- ILE-HIS-MET-THR-GLU-PHE-LEU-ASP-LYS-LEU-ASP-PHE- residues 65-96 of carboxypeptidase A is discussed in ref. 3. GLY -GLN-ASN-PRO-SER-PHE-THR-ALA- I LE-LEU-ASP-SER-MET-ASP- I LE- Residues 93-118: Derived from sequence analysis of the 120 "heavy" chain of Fractions I and II (Fig. 3). The overlap of TYR-VAL-LEU-PR0-VAL-VAL-ASN-ILE-ASP-GLY-TYR-ILE-TYR- residues 93-96 with the carboxyl terminus of "CN-2" aids in the PHE-LEU-GLU- ILE-VAL-THR-ASN-PRO-ASN-GLY-PHE-ALA-PHE-THR-HIS- alignment. Residues 246-257: Derived from the sequence of a peptide labeled at the active site of carboxypeptidase B (13) and aligned both by structural homology and by the functional analogy of 255 THR-ILE-TYR-PRO-ALA-SER-GLY-GLY-SER-ASP- Tyr-248. GLY-SER-ILE-ILE-THR-THR-ILE-TYR-GLN-ALA-SER-GLY-GLY-SER-ILE- Residues 280-293: Derived from the sequence of the sole cysteinyl peptide in carboxypeptidase B isolated by Winters- 270 ASP-TRP berger (14) and aligned by structural homology (3). Residues 294-308: Derived from the sequence of the carboxyl ASP-TRP-SER-TYR-ASN-GLN-GLY-ILE-LYS-TYR-SER-PHE-THR-PHE-GLU- terminal peptide "CN-1" of Elzinga et al. (15) assuming methio- 285 nine as the site of cyanogen bromide cleavage. Its placement ad- (TYR,GLY ,PHE)VAL-LEU-PRO-GLU-SER-GLN- jacent to residue 293 is supported by the composition of a tryptic LEU-ARG-ASP-THR-GLY-ARG-TYR-GLy-PHE-LEU-LEU-PRO-ALA-SER-GLN- peptide, "T-28" (16), which would correspond to residues 277- 300 298 and extend a possible sequence back from 279 to 277, as ILE-GLN-PRO-THR-CYS-GLU-GLU-THR-MET-LEU-ALA-ILE-LYS-TYR-VAL- indicated in parentheses. I LE- ILE-PRO-THR-ALA-GLN-GLU-THR-TRP-LEU-GLY-VAL-LEU-THR-I LE- 307 THR-SER-TYR-VAL-LEU-GLU-HIS-LEU between two peaks, subsequently called Fraction I and Frac- MET-GLU-HIS-THR- VALASN-ASNLE-U tion II. Sequence analyses of the amino terminal regions of the purified enzyme and its component chains were performed with a Beckman Sequencer following the general procedure of dases were eluted from this. column by application of 0.02 M Tris, pH 7.5, containing 0.5 M NaCI, whereas trypsin and Edman and Begg (6). A volatile buffer system was employed, and 1 ,4-butanedithiol was added to the chlorobutane to chymotrypsin remained adsorbed. The column could be re- generated by elution of the endopeptidase activities with 0.1 stabilize the sequenator products (7). M sodium acetate, pH 3.0. The carboxypeptidase fractions RESULTS AND DISCUSSION were concentrated to 220 ml in a Diaflo apparatus (UM-10 Homogeneity membrane) and were subsequently dialyzed against 0.02 M Tris, pH 7.5, for 36 hr. Under these conditions, 94% of car- Fractions I and II were almost pure, as judged by analytical boxypeptidase A but only 2.5% of carboxypeptidase B pre- polyacrylamide disc gel electrophoresis (Fig. 2). Furthermore, cipitated. each of the two fractions was distinct from carboxypeptidase The supernatant solution was applied to a 2.5 X 35 cm B prepared from procarboxypeptidase B, which migrated column of E-aminocaproyl-D-tryptophan-Sepharose, prepared with a lower Rf than either of the carboxypeptidases B isolated by covalent coupling of E-aminocaproyl-D-tryptophan to from active pancreatic juice. Sepharose 4B activated with cyanogen bromide (5). The re- Fractions I and II were also examined by electrophoresis in sults of this chromatography are shown in Fig. 1 (A and B). polyacrylamide gels in the presence of sodium dodecyl sulfate Both carboxypeptidases were retarded by the modified and ,3-mercaptoethanol (9) (Fig. 2). Each enzyme displayed Sepharose. Carboxypeptidase B was eluted by 0.15 M NaCl, two predominant protein bands in this system, corresponding while the elution of carboxypeptidase A required a higher salt to molecular weights of approximately 10,000 and 25,000. concentration. The purity of carboxypeptidase B thus pre- Native carboxypeptidase B displayed a single band corre- pared varied from 70 to 90% as judged by specific activity sponding to a molecular weight of approximately 34,000, toward benzoylglycyl-Larginine (1). Final purification was which is very close to that previously reported for the enzyme effected by chromatography on DE-52 cellulose, as shown in (1). We conclude, therefore, that each enzyme isolated from Fig. 1 (C and D). Carboxypeptidase B activity was distributed activated pancreatic juice contains a break in the chain ap- Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 68 (1971)" Structure of Carboxypeptidases A and B 1229

FIG. 3. Identification on the model of bovine carboxypeptidase A of the sites of internal cleavage in bovine carboxypeptidase B isolated from spontaneously activated pancreatic juice. Bond cleavage in Fraction I occurred at residues 95-96 and in Fraction II at residues 92-93. This illustration has been adapted from Lipscomb et at. (17).

proximately 100 residues from one end. The two fragments of Assuming that the three-dimensional structures of car- molecular weight 10,000 and 25,000 are subsequently referred boxypeptidases A and B are similar (3), one can examine the to as "light" and "heavy" chains, respectively. position of the bond cleavages in carboxypeptidase B on the three-dimensional model of A The Location of sites of limited proteolysis carboxypeptidase (17). two bonds, residues 92-93 and 95-96, occur on an exposed loop of Since amino terminal residues of native carboxypeptidase B the chain on the surface of the model (Fig. 5) and their (10) and of the light chains of Fractions I and II are threonine, locations are consistent with the assumption of conforma- the light chain (Fig. 3) appears to correspond to the amino tional homology. terminal region of the intact enzyme. The amino acid composition of the light chain indicates a chain length of 95-98 Structural relationships of carboxypeptidases B and A residues. Precise positions can be assigned to the sites of chain Sequenator analysis of carboxypeptidase B has identified the cleavage on the basis of homology with carboxypeptidase A. sequence of the first 30 amino terminal residues as well as an Elzinga and Hirs (11) have isolated a peptide "CN-2" from interior 26-residue sequence of the enzyme beginning with carboxypeptidase B which, by homology with carboxy- residue 93. Together with previous sequence data, this new peptidase A, can be placed at residues 65-96 (3). If the pro- information has extended the sequence comparison by 51 posed order of the light and heavy chains is correct, the car- residues and has strengthened the hypothesis of sequence boxyl terminus of CN-2 (-Gly-Arg-Glu-Ile-His-MNet) homology between bovine carboxypeptidases A and B. should correspond to the amino terminus of the heavy Specific residues at homologous loci are compared in Table chain. As shown in Fig. 3, the amino terminus of the heavy 1. On the basis of the three-dimensional model of Lipscomb chain of Fraction II does begin with a sequence identical with et al. (18), side chains in carboxypeptidase A were tentatively the C-terminal tetrapeptide of CN-2. Since, in addition, the defined as occupying either surface or interior positions. Of heavv chain of Fraction II overlaps the heavy chain of Frac- the 125 residues compared, 47 were identical in carboxy- tion I and CN-2, it is possible to assemble the structure from peptidases A and B. Of the 78 replacements, 25 preserve their residues 64 to 118, as shown in Fig. 4. polar character and 29 preserve their hydrophobic character; Downloaded by guest on September 26, 2021 1230 Biochemistry: Reeck et al. Proc. Nat. Acad. Sci. USA 68 (1971)

many of the latter are conservative substitutions. Twenty-four the hypothesis of conformational homology is at position 255, replacements involve a distinct change in side-chain character which is occupied by isoleucine in carboxypeptidase A and between the two enzymes. Sixteen of these are on the surface by aspartic acid in carboxypeptidase B. However, as discussed of carboxypeptidase A and presumably are less important for above, this residue is suitably located to provide the negative conformational integrity than the remaining eight "interior" charge in the binding site of carboxypeptidase B. residues. Among the latter, four of the five replacements of interior polar residues in carboxypeptidase A involve little This work was supported in part by research grants from the change in side-chain volume (Table 1). Perhaps the most in- National Institutes of Health (G1I 15731) and the American teresting change is found at position 255, which is occupied by Cancer Society (P-79M). G.R.R. has been a recipient of a pre- isoleucine in the hydrophobic substrate-binding pocket of doctoral fellowship of the National Science Foundation. carboxypeptidase A and by aspartic acid in carboxypeptidase B. If, as postulated, the two enzymes possess conformational 1. Wintersberger, E., 1). J. Cox, and H. Neurath, Biochemistry, is in to the anionic 1, 1069 (1962). homology, aspartic acid a position provide 2. Neurath, H., K. A. Walsh, and W. P. Winter, Science, 158, binding site of cationic substrates for carboxypeptidase B. 1638 (1967). This situation would parallel the difference between chymo- 3. Bradshaw, R. A., H. Neurath, and K. A. Walsh, Proc. Nat. trypsin and trypsin (19), where an analogous change in speci- Acad. Sci. USA, 63, 406 (1969). ficity is due to the difference between Ser-189 in chymotrypsin 4. Keller, P. J., E. Cohen, and H. Neurath, J. Biol. Chem., 233, 344 (1958). and Asp-177 in trypsin (20, 21). 5. Folhkwing the general procedures of Cuatrecasas, P., M. The chain cleavage in carboxypeptidase B has proved use- Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. USA, 61, ful in extending the sequence analysis of this enzyme. It is well 636 (1968); Axon, R., J. Porath, and S. Ernback, Nature, known that proteolysis (22) occurs in vivo (e.g., zymogen 214, 1302 (1967); and Porath, J., R. Ax~n, and S. Ernback, of as well Nature, 215, 1491 (1967). activation, blood clotting, assembly bacteriophage) 6. Edman, P., and G. Begg, Eur. J. Biochem., 1, 80 (1967). as in vitro (e.g., formation of plakalbumin, ribonuclease-S). 7. Hermodson, M. A., L. H. Ericsson, and K. A. Walsh, Fed. Thus, as a general method, experimental conditions might be Proc., 29, 728 (1970). selected for the proteolytic cleavage of a single peptide bond in 8. Davis, B., Ann. N.Y. Acad. Sci., 121, 404 (1964). a protein. This cleavage point would then form the starting 9. Weber, K., and M. Osborn, J. Biol. Chem., 244, 4406 (1969). 10. Ccx, D. J., E. Wintersberger, and H. Neurath, Biochemistry, point for sequenator analysis. The effectiveness of this method 1, 1078 (1962). would depend upon the specificity of the protease and upon 11. Elzinga, M., and C. H. W. Hirs, Arch. Biochem. Biophys., the conformational characteristics favoring unique bond 123, 361 (1968). cleavage in the protein substrate. 12. Bradshaw, R. A., L. H. Ericsson, K. A. Walsh, and H. Neurath, Proc. Nat. Acad. Sci. USA, 63, 1389 (1969). CONCLUSIONS 13. Plummer, T. H., Jr., J. Biol. Chem., 244, 5246 (1969). 14. Wintersberger, E., Biochemistry, 4, 1533 (1965). The following new observations are consistent with homology 15. Elzinga, M., C. Y. Lai, and C. H. W. Hirs, Arch. Biochem. of carboxypeptidases A and B at the level of three-dimensional Biophys., 123, 353 (1968). conformation: 16. Elzinga, M., and C. H. W. Hirs, Arch. Biochem. Biophys., 1. The sites of bond cleavage in Fractions I and II of the 123, 343 (1968). 17. Lipscomb, W. N., J. A. Hartsuck, F. A. Quiocho, and G. N. newly isolated carboxypeptidases B are at positions corre- Reeke, Jr., Proc. Nat. Acad. Sci. USA, 64, 28 (1969). sponding to an exposed loop on the surface of carboxypeptidase 18. Lipscomb, W. N., J. A. Hartsuck, G. N. Reeke, Jr., F. A. A. Quiocho, M. L. Ludwig, T. A. Steitz, P. Bethge, H. Muir- 2. Of the 125 residues that have been compared (Table 1), head, and J. C. Coppola, Brookhaven Symp. Biol., 21, 24 in A. (1968). 78 are exposed to the solvent carboxypeptidase Sixty- 19. Wa.lsh, K. A., and H. Neurath, Proc. Nat. Acad. Sci. USA, two of these are either identical in carboxypeptidase B or have 52, 884 (1964). polar characteristics. Among the 47 "interior" residues, 43 are 20. Steitz, T. A., R. Henderson, and D. -M. Blow, J. Mol. Biol., either identical in carboxypeptidase B or have retained their 46, 337 (1969). hydrophobic characteristics. Thus the pattern of residues 21. Walsh, K. A., L. L. Houston, and R. A. Kenner, in Struc- ture-Function Relationships of Proteolytic Enzymes, ed. P. forming the hydrophobic interior of carboxypeptidase A is Desnuelle, H. Neurath, and M. Ottesen (Munskgaard, largely preserved in the sequence of carboxypeptidase B. In Copenhagen, 1970), p. 56. fact, the only residue replacement apparently at variance with 22. Ottesen, M., Annu. Rev. Biochem., 36, 55 (1968). Downloaded by guest on September 26, 2021