Proc. Nat. Acad. Sci. USA Vol. 72, No. 5, pp. 1666-1670, May 1975

Amino-Acid Sequence of Bovine B (pancreatic juice/substrate specificity/homology) KOITI TITANI, LOWELL H. ERICSSON, KENNETH A. WALSH, AND HANS NEURATH Department of Biochemistry, University of Washington, Seattle, Wash. 98195 Contributed by Hans Neurath, February 24, 1975

ABSTRACT The amino-acid sequence of bovine car- ods of Gross and Witkop (15) and Omenn et al. (16), respec- boxypeptidase B [peptidyl-L-lysine(-L-arginine), tively. Digestions of polypeptides with TPCK-trypsin EC 3.4.12.3] has been determined using the heavy and light chains of the isolated from spontaneously (Worthington), a-chymotrypsin [Worthington treated with activated pancreatic juice. Comparison of the sequence TLCK (17)1, and thermolysin (Daiwa K.K.) were carried out with that of shows that the two en- in the pH-stat at pH 8.0 and 370 using a 1: 50 molar ratio of zymes are homologous (49% identity) and that all but one enzyme to substrate. Prior to tryptic digestion some poly- of the functional residues identified in carboxypeptidase by the method of Yaoi et al. (18). A occur in corresponding loci in (pep- peptides were succinylated tidyl-L- hydrolase, EC 3.4.12.2). The exception Mixtures of large peptides were separated on columns of is the replacement of Ile-255 at the bottom of the sub- Sephadex G-50 in 9% formic acid or 0.1 M ammonium bi- strate binding pocket of carboxypeptidase A, by aspartic carbonate, and further purified, if necessary on a column of acid in carboxypeptidase B. This single change can ac- at by applying a gradient of 0.1 M two . SE-Sephadex C-25 500 count for the difference in specificity of the sodium formate, pH 3.1, to 2.0 M sodium formate, pH 5.0, Bovine carboxypeptidase B [peptidyl-L-lysine(-L-arginine)- in the presence of 7 M urea. Small peptides were primarily hydrolase, EC 3.4.12.3], isolated from partially purified pro- fractionated on a Dowex 1 X2 column with pyridine acetate carboxypeptidase B (1, 2) is composed of a single polypeptide buffers (19), and heterogeneous fractionswere further separated chain, and resembles carboxypeptidase A (peptidyl-L-amino on a Dowex 50 X2 column with pyridine acetate buffers (20). acid hydrolase, EC 3.4.12.2) in molecular weight, amino-acid Amino-acid composition was determined on the Spinco composition, metal content, and mechanism of action. It model 120 Amino-Acid Analyzer according to the method of differs from carboxypeptidase A in its substrate specificity Spackman et al. (21). Sequenator analysis was performed with which is directed towards basic rather than hydrophobic a Beckman Sequencer model 890 by a modification (22) of residues. Bradshaw et al. (3) proposed that the two enzymes the method of Edman and Begg (23). Disc gel electrophoresis have homologous amino-acid sequences and similar three- in the presence of sodium dodecyl sulfate was performed by the dimensional structures. This hypothesis has been strengthened method of Weber and Osborn (24). Subtractive Edman by partial sequence data from our laboratory and others degradations were carried out by a modification of the method (4-11). of Konigsberg and Hill (25). Digestions with carboxypepti- Reeck et al. (4, 12) isolated two new forms of carboxypepti- dases A and B, and yeast carboxypeptidase C were performed dase B from spontaneously activated pancreatic juice and as described by Ambler (26) and Hermodson et al. (27). showed that they are derived by two alternate internal splits RESULTS in the parent single-chain form. Each of the new forms (CPB-I and CPB-II) could be separated into two chains. This proce- The amino-acid sequences of the light and the heavy poly- placement of peptide chains of B-I and B-II were deter- dure facilitated sequence analysis and permitted intact all of the residues in the sequence of the enzyme. The comple- mined largely by automatic sequence analysis of the tion of the amino-acid sequence of the enzyme has aided the chains and of fragments produced by chemical or enzymatic and has cleavage of methionyl, tryptophanyl, and arginyl bonds as refinement of the model derived by x-ray analysis* of enabled a detailed examination of the structural homology of summarized in Table 1 and Fig. 1. The remaining portions carboxypeptidases A and B. fragments and the residues not clearly identified by sequenator analysis were resolved by conventional methods. In Fig. 2, MATERIALS AND METHODS the complete sequence is shown and compared to that of Isolation of carboxypeptidases B-I and B-Il, and subsequent carboxypeptidase A. For ease of comparison, the numbering separation of the component light and heavy chains was of residues in carboxypeptidase A is used for carboxypeptidase carried out according to the method of Reeck et al. (12). B. and of polypeptides Sequenator analysis of the whole light chain yielded the S-Aminoethylation S-pyridylethylation for were performed by the methods of Raftery and Cole (13) and amino-terminal sequence from Thr4 to Asn-45 except Friedman et al. (14), respectively. Cleavages of polypeptides at residues 34, 36, and 43 (Table 1), which were subsequently methionine with cyanogen bromide and at tryptophan with identified on fragments. The remainder of the sequence of the BNPS-skatole were accomplished by adaptation of the meth- light chain was determined on two cyanogen bromide frag- ments CBi and CBii, 11 chymotryptic peptides of the S- * J. R. Herriott and M. F. Schmid, in preparation. aminoethylated light chain, nine peptides derived by tryptic 1666 Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Bovine Carboxypeptidase B 1667 TABLE 1. Fragments prepared for sequence analyses of carboxypeptidase B Sequenator analysis No. of Residue degrada- Residues Tentative Fragment* Method of preparation no.t tions identified identification Li Light chain of CPB-It 4-89 Lii Light chain of CPB-IIt 4-89 42 4-45 34, 36, 43 CBi Cleavage of Li or LzI with CNBr 4-64 CB,, Cleavage of Li or Lii with CNBr 65-89 10 65-74 Hi Heavy chain of CPB-It 96-308 31 96-126 119, 121, 122 HII Heavy chain of CPB-IIt 93-308 19 93-111 CBiii Cleavage of Hii with CNBr 93-96 CBiv Cleavage of HI or Hii with CNBr 97-125 CBv Cleavage of HI or HII with CNBr 126-201 30 126-155 143, 151, 152, (or 202)§ 153, 154 CBv-Ts-5 Cleavage of succinylated CBv with trypsin 146-184 26 146-171 168 CBv-Ts-6 Cleavage of succinylated CBv with trypsin 185-201 (or 202)§ CBv-Trp-3 Cleavage of CBv with BNPS-skatole 152-201 22 152-173 157, 162, 164, (or 202)§ 166, 168, 172 CBvi Cleavage of HI or Hil with CNBr 202-294 51 203-253 236, 237, 239, (or 203)§ 245, 246 CBvi-Trp-2 Cleavage of CBvi with BNPS-skatole 258-294 29 258-286 283, 284, 285 CBvil Cleavage of HI or HIl with CNBr 295-308

* For identification, see also Fig. 1 and the text. t Residue numbers correspond to those of carboxypeptidase A in Fig. 2. t These fragments are described by Reeck et al. (4) who also report the sequences corresponding to residues 4-33 of LII and of residues 96-118 of HI. § These ambiguities in residue numbers are caused by the presence of the two adjacent methionine residues at positions 201 and 202. See the text.

digestion of CB, and CBI1, and six subpeptides of a tryptic sible because of the low yield (approximately 10-15%) of the peptide (residues 36-64) obtained by digestion with ther- corresponding cyanogen bromide fragment CBiv (residues molysin. 97-125) and the failure to isolate smaller peptides from this The results indicated that the carboxyl-terminal residue of portion of the molecule. However, comparison of the amino- the light chain of the CPB-I is Thr-89, not His-95 as pre- acid composition of CBiv with the sequenator analysis of viously predicted (4). The sequence of a fragment, "CN-2", the heavy chains indicates that all three residues must be reported by Elzinga and Hirs (8), corresponds to residues threonine. 65-96, ending with the sequence -Valv-Arg-Thr-Tyr-Gly-Arg- Since the cyanogen bromide fragment CBv is the only one Glu-Ile-His-Met96. As pointed out by Reeck et al. (4), this containing an amino-terminal tryptophan, it was placed fol- sequence provides an overlap of the carboxyl-terminus of the lowing Met-125 by virtue of an overlap provided by the light chains of CPB-I and CPB-II with the corresponding sequenator analysis of the whole heavy chain HI. Sequenator heavy chains. The heavy chain of CPB-II begins with Glu93- analysis of fragment CBv yielded a sequence from Trp-126 Ile-His-Met-Thr-; that of CPB-I begins with Metw-Thr-. through Gly-155 (Table 1). A large tryptic peptide, CBv-TS-5, Since the light chain of CPB-I ends with the carboxyl- isolated from succinylated fragment CBv, yielded the sequence terminal sequence -Val-Arg-Thr89 and the heavy chain begins from Asn-146 to Glu-171 (Table 1). Sequenator analysis of with Met96, this form of the enzyme lacks the pentapeptide fragment CBv-Trp-3, isolated by cleavage of the fragment corresponding to residues 90-95. It is probable that limited with BNPS-skatole, extended the sequence by only two tryptic or chymotryptic cleavage at Arg-92 or His-95 occurred residues up to Glu-173 and confirmed the sequence obtained in the crude pancreatic juice and that the carboxyl-terminal for fragment CBv-Ts-5 (Table 1). In these analyses, residues portions of the newly generated light chains were subsequently 168 and 172 were only tentatively identified as serine. Positive removed by carboxypeptidases A and B. The extent of this identifications of- these residues and further extension of the removal is established in CPB-I but not in CPB-II. However, sequence were obtained from the compositions and sequences x-ray crystallographic data* indicate that the carboxyl- of three tryptic subpeptides of a chymotryptic peptide (resi- terminus of the light chain of CPB-II is either on the surface dues 166-182) isolated from the whole heavy chain HiI. The of the molecule and in a disordered state or is lacking residues remainder of the sequence of fragment CBv was determined 86-92. by conventional methods using four chymotryptic peptides of Combined sequenator analyses of the heavy chains (HI and the whole heavy chain HI,. These peptides were aligned with HII) yielded a sequence from Glu-93 to Trp-126 and tenta- the aid of the partial sequence of peptide CBv-Ts-6 isolated tively identified residues 119, 121, and 122 (Table 1). Un- from a tryptic digest of succinylated fragment CBv (Table 1). equivocal identification of these three residues was not pos- The sequence of one of the chymotryptic peptides isolated Downloaded by guest on October 1, 2021 1668 Biochemistry: Titani et al. Proc. Nat. Acad. Sci. USA 72 (1975)

.4 50 100 150 290 250 3QI8 Carboxypeptidose B Li -I Carboxypeptidose B-I iS//T I/f s ~~~~~~~H.,

CBI E CB//I CBVI C CBII CB/ ITrypsin after Skotoe CBYU S uccinylation- SkatoleBNPS- / mm CBysT-5D CBy1-Trp-2 CByTs-6 1 CBy-Trp-3 Elzinga et GI.. (7, 8) Wintersberger (6) CN-2 DCN-I Plummer (10) Kimmel a Plummer (II)

FIG. 1. Schematic representation of the alignment of fragments used for the determination of the amino acid sequence of carboxypep- tidase B. Fragment Li and HI are products of autolysis of carboxypeptidase B-I and were isolated after reduction and alkylation of the disulfide bonds of the enzyme (4). Fragments CBI through CBvii were generated by cleavage with CNBr. Subfragmentations of CBv and CBvi were effected with either BNPS-skatole at tryptophanyl residues (CBv-Trp-3 and CBvi-Trp-2) or with trypsin at arginyl residues (CBv-Ts-5 and CBv-Ts-6). Fragments obtained by other authors (e.g., CN-1 and CN-2) are shown at the bottom (6-8, 10, 11). The cross- hatched areas denote sequences determined with the Sequencer. For simplicity, only carboxypeptidase B-I is illustrated. The specific sequencer analysis ascribed to the light chain of B-I was in fact performed on the light chain of B-II (see Table 1). For further details see the text and Table 1.

from the whole heavy chain Hil was Ser-Ser-Ile-Lys-Ala-Tyr a sequence from Ala-258 to Ile-286 with poor identifications and corresponded to residues 188-192 (Fig. 2). This result of residues 283-285. These identifications were confirmed and indicated that carboxypeptidase B has one extra residue be- the remainder of the sequence of fragment CBvj resolved by tween residues 186 and 190 when compared with carboxy- conventional methods using 13 tryptic peptides of the frag- peptidase A. The insertion of this residue in this region is ment. The sequence of fragment CBvi includes the functional supported by the x-ray data* and is also consistent with the residues Tyr-248 (10), Glu-270 (5, 11), and the sole sulfhydryl composition of the tryptic peptide AE-T-21 of Elzinga and group (6) of the enzyme. Hirs (28). The cyanogen bromide fragment CBvii was placed in the The verification of two adjacent methionine residues at carboxyl-terminal portion of the heavy chain because it lacked positions 201 and 202 presented problems. Repeated amino- homoserine. The sequence of this fragment was determined by acid analyses of fragments CBv and CBvI showed that both conventional methods using the intact fragment and three fragments contained more than one residue of homoserine tryptic peptides. This sequence is identical to that of fragment (approximately 1.2-1.4) per mole. Whereas sequenator analy- "CN-1" of Elzinga et al. (7). sis of fragment CBv yielded a single amino-terminal sequence, analysis of fragment CBvI always yielded two simultaneous DISCUSSION sequences of Leu-Tyr-Pro-Tyr- and X-Leu-Tyr-Pro- in a The amino-acid sequence of bovine carboxypeptidase B ratio of 8:1. These results suggested the presence of two described herein is based on present data and on those reported adjacent methionine residues. The amino-acid compositions in previous communications from this laboratory (3-6), except of the single-chain enzyme (2) and the two-chain enzyme (12) for residues 90-92 which are derived from fragment CN-2 of provide some indirect support for this conclusion since both Elzinga and Hirs (8). This sequence is consistent with the enzymes contain six methionine residues and only four of composition and sequence of fragments of the enzyme pub- them are placed at residues 64, 96, 125, and 294. The presence lished by other investigators (7-11) and with the summary of of two methionine residues at 201 and 202 is consistent with partial sequences recently reported by Schmidt and Hirs (9). the x-ray analysis.* The pairing of one- of the three disulfide bonds (between Sequenator analysis of fragment CBvi yielded a sequence residues 66 and 79) has been previously described (8); the from Leu-203 to Gly-253 except for tentative identifications other two have been placed between residues 138 and 161 and of residues 236, 237, 239, 245, and 246 (Table 1), suggesting between residues 152 and 166 by x-ray crystallographic that these residues are either threonine or serine. Another analysis (29). sequenator analysis of fragment CBvy-Trp-2 (Table 1) yielded Comparison of the amino-acid sequence of carboxypepti- Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Bovine Carboxypeptidase B 1669

5 10 15 20 25 30 Carboxypeptidase B Thr-Thr-Gly-Hi s-Ser-Tyr-Gl u-Lys-Tyr-Asn-Asn-Trp-GJ u-Thr-Ile-Glu-A1la-Trp-Thr-GJlu-Gl n-Val -Al a-Se'r-Gl u-Asn-Pro - Carboxypeptidase A AAa-Arg-Ser-Thr-Asn-Thr-Phe-Asn-Tyr-Ala-Thr-Tyr-His-Thr-Leu-Asp-Glu-Ile-Tyr-Asp-Phe-Met-Asp-Leu-Leu-Val-A1a-Glu-His-Pro-

35 40 45 50 55 60 65 Asp-Leu- Il e-Ser-Arg-Ser-Ala-Il e-Gly-Thr-Thr-Phe-Leu-Gly-Asn-Thr-I 1e-Tyr-Leu-Leu-Lys-Val -Gly-Lys-Pro-Gly-Ser-Asn-Lys-Pro-Ala-Val -Phe-Met-Asp- G1n-Leu -Val -Ser-Lys-Leu-Gl n-Il e-Gly-Arg-Ser-Tyr-Glu-Gly-Arg-Pro- I le-Tyr-Val -Leu-Lys-Phe-Ser-Thr-Gly-Gly-Ser-Asn-Arg-Pro-Ala-I 1e-Trp-I 1e-Asp-

70 75 80 85 90 95 100 Cys-Gly-Phe-Hi s-Al a-Arg-Gl u-Trp-Il e-Ser-Pro-Al a-Phe-Cys-Gl n-Trp-Phe-Val -Arg-Glu-Al a-Val -Arg-Thr-Tyr-Gly-Arg-Glu-Il e-Hi s-Met-Thr-Glu-Phe-Leu- Leu-Gly-Il e-His-Ser-Arg-Glu-Trp-Ile-Thr-Gl n-Al a-Thr-Gly-Val -Trp-Phe-Al a-Lys-Lys-Phe-Thr-Gl u-Asn-Tyr-Gly-Gl n-Asn-Pro-Ser-Phe-Thr-Al a-I 1e-Leu-

105 110 115 120 125 130 135 Asp-Lys-Leu-Asp-Phe-Tyr-Val -Leu-Pro-Val -Val -Asn-I 1e-Asp-Gly-Tyr- Il e-Tyr-Thr-Trp-Thr-Thr-Asn-Arg-Met-Trp-Arg-Lys-Thr-Ar-Ser-Thr-Arg-Ala-Gly- Asp-Ser-Met-Asp-I 1e-Phe-Leu-Gl u-Il e-Val -Thr-Asn-Pro-Asn-Gly-Phe-Ala-Phe-Thr-Hi s-Ser-Glu-Asn-Arg-Leu-Trp-Arg-Lys-Thr-Arg-Ser-Val -Thr-Ser-Ser-

140 145 lO 155 160 165 170 Ser-Ser-Cys-Thr-Gly-Thr-Asp-Leu-Asn-Arg-Asn-Phe-Asp-Al a-Gly-Trp-Cys-Ser-I le-Gly-Al a-Ser-Asn-Asn-Pro-Cys-Ser-Gl u-Thr-Tyr-Cys-Gly-Ser-Ala-Ala- Ser-Leu-Cys-Val -Gly-Val -Asp-Al a-Asn-Arg-Asn-Trp-Asp-Al a-Gly-Phe-Gly-Lys-Al a-Gly-Ala-Ser-Ser-Ser-Pro-Cys-Ser-Gl u-Thr-Tyr-Hi s-Gly-Lys-Tyr-Al a-

175 180 185 190 195 200 205 Glu-Ser-Glu-Lys-Glu-Ser-Lys-Ala-Val-Al a-Asp-Phe-I le-Arg-Asn-Hi s-Leu-Ser-Ser- 11e-Lys-Ai a-Tyr-Leu-Thr-I le-His-Ser-Tyr-Ser-Gl n-Met-Met-Leu-Tyr-Pro- Ile As n-Ser-Gl u-Val -Gi u-Val -Lys-Ser- W -Val -Asp-Phe-Val -Lys-Asn-Hi s-Gly-Asn- -Phe-Lys-Al a-Phe-Leu-Ser-I 1 e-His-Ser-Tyr-Ser-Gl n-Leu-Leu-Leu-Tyr-Pro-

210 215 220 225 230 235 240 Tyr-Ser-Tyr-Asp-Tyr-Lys-Leu-Pro-Lys-Asn-Asn-Val -Glu-Leu-Asn-Thr-Leu-Al a-Lys-Gly-Al a-Val -Lys-Lys-Leu-Al a-Ser-Leu-His-Gly-Thr-Thr-Tyr-Ser-Tyr- Tyr-Gly-Tyr-Thr-Thr-Gl n-Ser- Il e-Pro-Asp-Lys-Thr-Glu-Leu-Asn-Gl n-Val -Al a-Lys-Ser-Al a-Val _A4~-Al a-Leu-Lys-Ser-Leu-Tyr-Gly-Thr-Ser-Tyr-Lys-Tyr-

245 250 255 260 265 270 275 Gly-Pro-Gly-Al a-Thr-Thr-I le-Tyr-Pro-Al a-Ser-Gly-Gly-Ser-Asp-Asp-Trp-Al a-Tyr-Asp-Gl n-Gly-I le-Lys-Tyr-Ser-Phe-Thr-Phe-Gl u-Leu-Arg-Asp-Lys-Gly- Gly-Ser- I 1e-I 1e-Thr-Thr- I1e-Tyr-G1n-Al a-Ser-Gly-Gly-Ser-Ile-Asp-Trp-Ser-Tyr-Asn-Gl n-Gly-I le-Lys-Tyr-Ser-Phe-Thr-Phe-Gl u-Leu-Arg-Asp-Thr-Gly-

280 285 290 295 300 305 Arg-Tyr-Gly-Phe-Val -Leu-Pro-Glu-Ser-Gl n-I le-Gl n-Pro-Thr-Cys-Glu-Glu-Thr-Met-Leu-Al a-I le-Lys-Tyr-Val -Thr-Ser-Tyr-Val -Leu-Giu-His-Leu Arg-Tyr-Gly-Phe-Leu-Leu-Pro-Ala-Ser-Gln-Ile-Ile-Pro-Thr-Ala-Gln-Glu-Thr-Trp-Leu-Gly-Val-Leu-Thr-Ile-Met-Glu-His-Thr-yVe-Asn-AsnLeu FIG. 2. Comparison of the amino-acid sequences of carboxypeptidase B (this work) and carboxypeptidase A (35). Residue numbers are those of carboxypeptidase A. Optimum alignment of the two sequences indicates that carboxypeptidase B is three amino acid residues shorter at the amino end than carboxypeptidase A, one residue longer at the carboxyl end, and has one residue inserted between residues 186 and 190. Residues 28 and 31 of carboxypeptidase A are glutamic acid and glutamine, respectively (36) rather than vice versa as re- ported by Bradshaw et al. (35). dases B and A confirms the earlier predictions of homology and Glu-72 (3), Glu-270, Tyr-248, Arg-145, and Tyr-198 based on partial sequence data, particularly those which (32, 33). The single exception is Ile-255 which in carboxy- include functionally or structurally significant amino-acid peptidase B is aspartic acid. The single thiol group in carboxy- residues (3, 4). Optimal alignment of the two sequences peptidase B is found in cysteine-290. It has no counterpart in required the assumption that carboxypeptidase B is lacking carbox-ypeptidase A and its role in the structure or function of the first three residues corresponding to carboxypeptidase A, carboxypeptidase B, if any, is obscure (34). extends one residue beyond the carboxyl-terminus, and has one additional residue inserted between positions 186 and 190. Our thanks are due to Drs. J. R. Herriott and Michael Schmid The degree of identity between these two polypeptide chains for keeping us informed on the progress of their crystallographic is This is of the same order as the identity between analyses (*, 30), and for giving us access to the model of carboxy- 49%. peptidase B in the course of this investigation. This collaboration bovine pancreatic trypsin and chymotrypsin A (40%), two has been of mutual benefit and has accelerated the progress of homologous enzymes which also differ primarily in side-chain research. We thank Dr. M. A. Hermodson for valuable assis- specificity (30, 31). In this connection it is noteworthy that tance in the sequenator analyses and discussions. We are also residue Ile-255, which in carboxypeptidase A is located at the indebted to Mrs. R. M. MacDonald, Mr. D. Grahn, Mrs. B. Moody, and Mr. R. R. Granberg for excellent technical assis- pit of the side-chain binding pocket, is replaced in carboxy- tance. This work was supported by grants from the National peptidase B by aspartic acid which serves to attract the Institutes of Health (GM 15731) and from the American Cancer positively charged side-chain of specific lysyl and arginyl Society (BC-91P). substrates. This situation is entirely analogous to the replace- ment of Ser-189 in chymotrypsin by aspartic acid in trypsin. 1. Wintersberger, E., Cox, D. J. & Neurath, H. (1962) Bio- With one exception, all functional residues which have been chemistry 1, 1069-1078. 2. Kycia, J. H., Elzinga, M., Alonzo, N. & Hirs, C. H. W. implicated as components of the of carboxypepti- (1968) Arch. Biochem. Biophys. 123, 336-342. dase A (32, 33) are preserved in carboxypeptidase B. These 3. Bradshaw, R. A., Neurath, H. & Walsh, K. A. (1969) Proc. include in particular the three Zn ligands, His-69, His-196, Nat. Acad. Sci. USA 63, 406-411. Downloaded by guest on October 1, 2021 1670 Biochemistry: Titani et al. Proc. Nat. Acad. Sci. USA 72 (1975)

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