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

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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 carboxypepti- dases 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 ho- mology, 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 electro- phoresis (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 chro- matography 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 resi- dues 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.
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