Amino-Acid Sequence of Bovine Carboxypeptidase B (Pancreatic Juice/Substrate Specificity/Homology) KOITI TITANI, LOWELL H
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Proc. Nat. Acad. Sci. USA Vol. 72, No. 5, pp. 1666-1670, May 1975 Amino-Acid Sequence of Bovine Carboxypeptidase 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)hydrolase, tively. Digestions of polypeptides with TPCK-trypsin EC 3.4.12.3] has been determined using the heavy and light chains of the enzyme 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 carboxypeptidase A 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 carboxypeptidase B (pep- peptides were succinylated tidyl-L-amino acid 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 enzymes. 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 carboxypeptidases 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).