2 Carboxypeptidase B

Carboxypeptidase B J . E. FOLK I . Introduction . . . . . . . . . . . A. Historical Background . . . . . . . B . Distribution I1. Purification of the Enzyme and Zymogen; Assay . . I11. Physical-Chemical Properties of Carboxypeptidaae B . A . Physical Properties . . . . . . . . B . Chemical Composition . . . . . . . C . End Groups and Amino Acid Sequences . . . D. Early Considerations of Homology between Bovine Carboxypeptidases A and B . . . . . IV . Physical-Chemical Properties and Activation of Procarboxypeptidase B . . . . . . . . . V . Enzymic Properties of Carboxypeptidase B . . . A . Specificity . . . . . . . . . . B . Kinetics and Competitive Inhibition . . . . C . Activation and Inhibition . . . . . . VI . Enzymic Properties of Metalloenzymes . . . . . VII . Comment on the Enzyme Mechanism . . . . . VIII . Use in Structural Analysis and Modification of Proteins and Peptides . . . . . . . . .

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67 57 59

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61 61 62 64

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67 69 69 71 75 76 77

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77

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1 Introduction

A . HISTORICAL BACKGROUND In 1931 Waldschmidt-Leit and co-workers (1) showed that extracts of porcine pancreas gland contained an enzyme that catalyzed the release

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1. E . Waldschmidt-Leitz. F . Ziegler. A . Schaffner. and L Wed. Z Physiol Chem 197. 219 (1931) . 57

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58

J. E. FOLK

of arginine from a variety of protamines. Their findings that an acylated protamine, the benzylidene derivative of clupein, served as a substrate, while protamine esters did not, led these workers to suggest that arginine was liberated by this enzyme from the COOH-terminal end of protamines. The name protaminase was chosen for this enzyme because it showed no hydrolytic action toward other proteins tested. Calvery ( 2 ) , however, found that egg albumin, although completely resistant to digestion by a protaminase preparation, could be rendered susceptible to this enzyme by first digesting it with proteases. He suggested that perhaps lysine and histidine, as well as arginine, were liberated by protaminase. During the ensuing 20 years, protaminase was generally considered to be identical with chymotrypsin (3, 4 ) , a belief based upon reports that protamine hydrolyzing activity could not be separated from proteinase activity ( 5 , 6 ) .A report in 1951 that 6X crystallized carboxypeptidase A (Chapter 1, this volume) catalyzed the hydrolysis of the protamine, salmine, raised the question of the possible identity of protaminase with carboxypeptidase A ( 7 ) . Two observations, namely, ( a ) that lysine was only slowly released from N-benzoylglycyl-L-lysine during incubation with high concentrations of a purified carboxypeptidase A preparation (8) and (b) that small amounts of an extract of commercial pancreas powder catalyzed the rapid release of lysine from this benzoyl dipeptide ( 9 ) , prompted a series of investigations that gave subsequent proof for the existence of a second carboxypeptidase, carboxypeptidase B, of pancreas tissue and served to identify it with the protaminase of Waldschmidt-Leitz and coworkers. Early reports (10,11) cited pronounced differences in the specificities of carboxypeptidases A and B and listed a series of specific competitive inhibitors for carboxypeptidase B. Carboxypeptidase B was found to act on peptides containing the basic amino acids lysine, arginine, 2. H. 0. Calvcry, JBC 102, 73 (1933). 3. J. B. Surnner and G. F. Somers, “Chemistry and Methods of Enzymes,” 2nd ed., p. 182. Academic Press, New York, 1947. 4. J. H. Northrop, M. Kunitz, and R. M. Herriott, “Crystalline Enzymes,” 2nd ed., p. 118. Columbia Univ. Press, New York, 1948. 5. J. H. Northrop, cited by K. Myrback in “Die Methoden der Fermentforschung” (E. Bamann and K . Myrback, eds.), p. 2029, Thieme, Leipzig, 1941. 6. R. A. Portis and K. I. Altman, JBC 169, 203 (1947).

7. Unpublished data cited in E. L. Smith, in “The Enzymes,” (J. B. Sumner and K. Myrback, eds.) 1st ed., Vol. 1, Part 2, p. 828, Academic Press, New York, 1951. 8. K . Hofmann and M. Bergmann, JBC 134, 225 (1940). 9. J. E. Folk, ABB 64, 6 (1956). 10. J. E. Folk, JACS 78, 3541 (1956). 11. J. E. Folk and J. A. Gladner, JBC 231, 379 (1958).

2.

CARBOXYPEPTIDASE B

69

ornithine, homoarginine, and S- (P-aminoethyl) cysteine as the COOHterminal group (11, 12). The well-defined preferential action of carboxypeptidase A toward COOH-terminal aromatic and branched chain aliphatic amino acids is detailed by Hartsuck and Lipscomb, Chapter 1, this volume. The identity of protaminase and carboxypeptidase B became e?ident from a report that a protaminase fraction, prepared essentially as originally described (I), catalyzed the release of lysine and S-(paminoethyl) cysteine, as well as arginine, from the COOH-terminal position of polypeptides (13). The early recognition that carboxypeptidase B has a specific precursor, procarboxypeptidase B, from which it is formed by tryptic activation (10,11), was confirmed by chromatographic separation of the zymogens of the two carboxypeptidases from fresh pancreatic secretions of the cow (14) and of the swine and dog (16). The action of carboxypeptidase B on polypeptides and proteins was shown t o be in complete accord with the specificity as predicted from studies on small synthetic substrates (16). As a result purified porcine carboxypeptidase B, because of its almost absolute specificity for COOHterminal basic amino acids ( 1 7 ) , has found wide use in end group analysis and, in conjunction with carboxypeptidase A, for sequence determination. In this regard the special value of carboxypeptidase B in releasing COOH-terminal lysine and arginine from peptides, derived by tryptic digestion of proteins, points up an early hypothesis that the particular physiological function of carboxypeptidase B is the first-step degradation of the products of tryptic digestion (11).

B . DISTRIBUTION Carboxypeptidase B, in the form of an inactive zymogen, probably occurs in the cellular secretions of the pancreas of most vertebrates. The proenzyme has been identified in extracts of the pancreas of the rat (18) as well as those of the dog (16), swine (16), and cow (14). The pancreatic enzyme and zymogen of the spiny Pacific dogfish have been isolated and partially characterized (19). 12. F. Tietze, J. A. Gladner, and J. E. Folk, BBA 26, 6.59 (1967). 13. L. Weil, T. S. Seibles, and M. Telka, ABB 79, 44 (1969). 14. P. J. Keller, E. Cohen, and H. Neurath, JBC 223, 467 (1956). 15. G. Marchis-Mouren, M. Charles, A. Ben Abdeljlil, and P. Desnuelle, BBA 50, 186 (1961). 16. J. A. Gldner and J. E. Folk, JBC 231, 393 (1958). 17. J. E. Folk, K . A. Piez, W. R. Carroll, and J. A. Gladner, JBC 235, a 7 2 (1960). 18. G. Marchis-Mouren, L. Padre, and P. Dewuelle, BBRC 13, 262 (1983). 19. J. W. Prahl and H. Neurath, Biochemistry 5, 4137 (1966).

60

J. E. FOLK

Activity toward carboxypeptidase B substrates has been detected in the body fluids urine (20, d l ) , blood plasma ( 2 2 ) , and lymph (ZO), in kidney cortex ( 2 3 ) , and in catheptic spleen preparations (24). Inhibitor studies indicate that the enzyme from swine blood serum is not identical t o swine pancreatic carboxypeptidase B ( 2 5 ) . An enzyme similar in specificity to carboxypeptidase B has been reported in the gastric juice of crayfishes (26). II. Purification of the Enzyme and Zymogen; Assay

Carboxypeptidase B may be isolated from aqueous extracts of acetone powder of autolyzed porcine pancreatic glands by a process involving fractionation with ammonium sulfate, followed by chromatography on DEAEcellulose (17, 2 7 ) . This swine enzyme has been obtained in crystalline form (27, 28). Bovine carboxypeptidase B may be readily crystallized following tryptic activation of either purified or partially purified proenzyme preparations (29, SO). The earliest attempts a t purification of the bovine proenzyme were made by selective extraction of the euglobulin precipitate obtained from aqueous extracts of acetone powders prepared from fresh pancreas glands ( 1 1 ) . The major impurity of these preparations, chymotrypsinogen B, was removed by chromatography on DEAE-cellulose ( 3 1 ) .The zymogen was subsequently isolated in pure form from extracts of acetone powders by the use of several ion exchange chromatographic steps ( 2 9 ) . Procarboxypeptidase B has also been purified chromatographically from 20. E. G. Erdos, E. M. Sloane, and I. M . Wohler, Bwchem. Pharmacol. 13, 893 (1964). 21. I. Innerfield, R. Harvey, F. Luongo, and E. Blincoe, Proc. SOC. Ezptl. Biol. M e d . 116, 573 (1964). 22. E. G. Erdos and E. M. Sloane, Biochem. Pharmacol. 11, 585 (1962). 23. I. Innerfield, F. S. Gimble, and E. Blincoe, Life Sci. 3, 267 (1964). 24. L. M. Greenbaum and R. Sherman, JBC 237, 1082 (1962); L. M. Greenbaum and K. Yamafuji, in “Hypertensive Peptides” (E. G. Erdos et al., eds.), p. 252. Springer, New York, 1966. 25. E. G. Erdos, H. Y . T. Yang, L. L. Tague, and N. Manning, Bwchem. Pharmacol. 16, 1287 (1967). 26. R. Kleine, 2. Vergleich. Physiol. 56, 142 (1967). 27. J. E. Folk, “Methods in Enzymology” (in press). 28. A. L. Baker, personal communication (1969). 29. E. Wintersberger, D. J. Cox, and H. Neurath, Biochemistry 1, 1069 (1962). 30. J. H. Kycia, M. Elzinga, N. Alonzo, and C. H. W. Him, ABB 123, 336 (1968). 31. J.-F. PechBre, G. H. Dixon, R. H. Maybury, and H. Neurath, JBC 233, 1364 ( 1958).

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61

CARBOXYPEPTIDASE B

fresh bovine pancreatic juice (30). The dogfish proenzyme has been isolated (19) by a procedure similar to that employed for the bovine zymogen (69). The most convenient and widely used assay for carboxypeptidase B employs spectrophotometric measurement of the release of arginine from hippuryl-L-arginine (1'7, 36). This procedure for rate measurement is based on the difference in ultraviolet absorbancy between N-benzoylamino acids and the corresponding carboxyl-substituted N-benzoylamino acids (3.3). It is applicable to a number of synthetic substrates for carboxypeptidase B and has been employed over a wide range of concentrations of substrates (32).In an alternate assay for carboxypeptidase B the ninhydrin procedure is used to measure arginine released from hippurylarginine ( 11) . 111. Physical-Chemical Properties of Carboxypeptidase B

A. PHYSICAL PROPERTIES Carboxypeptidase B preparations from several species have been studied in some detail. Pertinent properties of these enzymes are listed in Table I. The swine enzyme appears homogeneous by several critieria: ion exchange chromatography at p H values 6 and 8, ultracentrifugation, diffusion, and moving boundary and polyacrylamide gel electrophoresis (17,3 4 ) . The mobility of the descending boundary a t 0" in moving cmz V-' sec-l boundary electrophoresis was found to be -2.38 x TABLE I MOLECULAR PROPERTIES OF CARBOXYPEPTIDASE B FROM SEVERALSPECIES Property

Swine

Molecular weight

34,300 3.24 S 8.16 X 10-7 ern* seeb1 0.720 cmag-I 1 g-atom/mole 21.4 at 278 nm

810.W D2O.W

V

Zinc E: Fm NHrterminal COOH-terminal

Threonine Threonine

cow 34,000 3.10 S

Dogfish 35,000-37,000 3.25 S

1 g-atom/mole 21.0 at 280 nm

Threonine Leucine

32. E. C. Wolff, E. W. Schirmer, and J. E. Folk, JBC 237, 3094 (1962). 33. G. W. Schwert and Y. Takenaka, BBA 16, 670 (1966). 34. J. E. Folk, unpublished results (1962).

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J. E. FOLK

in 0.05M potassium phosphate buffer, p H 7.02 (17).No study of the influence of pH and ionic strength on electrophoretic mobility has been conducted. The behavior of the enzyme on anion exchange cellulose under various conditions of pH, however, would suggest that the isoelectric point of the enzyme at low ionic strength (<0.1) is not far below 6.0 (27, 34). A molecular weight of 34,300 was calculated by the use of the sedimentation constant, s ~ , , , ~the , diffusion constant, Deo,w and the pycnometrically determined partial specific volume, recorded in Table I (17).A of 0.73 cm3 g1has been calculated from the amino acid composition ( 1 7 ) . The molecular weight of the bovine enzyme (29)and that of the dogfish (19) recorded in Table I were determined by short column sedimentation equilibrium assuming values for 7 of 0.730 and 0.735 cm3 g-', respectively. The sedimentation pattern (29) and polyacrylamide gel electrophoretic pattern (30) of the crystalline bovine enzyme were found to be consistent with homogeneity. The variations in the published values for the apparent weight-average molecular weights and the z-average molecular weights of the dogfish enzyme (19) indicate some degree of heterogeneity. Concentrated solutions of swine carboxypeptidase B in dilute tris buffer, pH 7.5,or suspensions of crystals of the enzyme in water have been stored frozen at -10" for periods up to 1 year without detectable loss in enzymic activity (27). Crystals of the bovine enzyme reportedly may be stored indefinitely as a suspension in 0.01 M tris HC1, p H 8.0, at 4" ($9). Studies with extracts of the pancreas of swine (36),cow (36),and rat (37) have shown the carboxypeptidase B of these species to be antigenically distinct from other hydrolases. The enzyme has been obtained as an immunochemically pure antigen from swine pancreas (38). An antiserum prepared against purified swine carboxypeptidase B (39)was found not to react with bovine carboxypeptidase B.

v

v,

B. CHEMICAL COMPOSITION Amino acid compositions of carboxypeptidase B preparations from the swine, cow, and dogfish are given in Table 11. A redetermination of the 35. J. Uriel and S. Avrameas, Ann. Znst. Pastew 106, 396 (1964). 36. S. Avrameas and J. Uriel, Protides Bwl. Fluids,Proc. Colloq. 12, 225 (1964). 37. J. Pascale, S. Avrameas, and J. Uriel, JBC 241, 3023 (1966). 38. S. Avrameas and J. Uriel, Biochemistry 4, 1750 (1965). 39. J. T. Barrett, Intern. Arch. Allergy Appl. Immunol. 26, 158 (1965).

2. CARBOXYPEPTIDASE B

63

composition of the bovine enzyme has shown one more residue each of aspartic acid and glycine, two more residues of proline, one less residue each of isoleucine and tyrosine, and three less residues of tryptophan (SO). This composition has recently been confirmed (41) with the exception that 12 residues of proline were found, in agreement with the earlier analysis (40).Bovine carboxypeptidase B contains one -SH group which is reactive to sulfhydryl reagents only after removal of the zinc atom from the enzyme (4.2). The remaining six half-cystine residues were shown to occur as three disulfide-bonded cystines (42).These findings, together with the initial report that bovine carboxypeptidase B contains 1 TABLE I1

AND DOGFISH CARBOXYPEPTIDASE B PREPARATIONS

AMINOACIDCOMPOSITIONS OF SWINE,Cow,

Amino acid

Swine" (Re~idues/34,300g)

Cow' (Residues/34,000 g)

Dogfishc (Residues/34,000 g)

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan Amide N

32.4 30.2 17.5 24.8 13.2 23.0 25.1 7.6 10.8 5.1 17.2 22.7 20.4 11.9 17.5 5.8 10.0 9.2 (27.8)

25.9 25.8 25.8 23.6 12.1 21.4 21.7 6.8 13.7 6.0 15.7 20.2 21.8 11.8 16.7 7.0 13.1 9.9 (22.9)

31.2 25.2 24.9 20.4 13.9 19.8 23.9

Total

304.4

299.0

4

7.2

16.8 8.2 19.6 16.9 19.6 9.2 14.6 4.1 13.8 9.6

-

298.9

From Folk et al. (17). From Cox et al. (40). From Prahl and Neurath (19).

40. D. J. Cox, E. Wintersberger, and H. Neurath, Biochemistry 1, 1078 (1962). 41. T. H. Plummer, Jr., JBC 244, 5246 (1969). 42. E. Wintersberger, H. Neurath, T. L. Coombs, and B. L. Vallee, Biochemistry 4, 1526 (1965).

64

J. E. FOLK

g-atom of zinc (40)) extend earlier observations that the swine enzyme, like swine (4.3) and bovine (44) carboxypeptidase A, contains 1 g-atom of tightly bound zinc (17)and that this metal serves as a structural and functional component of the enzyme (17 , 46). Similarities in the composition of porcine, bovine, and dogfish carboxypeptidases B, together with their general likeness in composition to bovine and porcine carboxypeptidases A (Chapter 1, this volume), except for the distribution of sulfur-containing amino acids, have prompted the suggestion of a common evolutionary origin for all of these exopeptidases (19). Recognition of small areas of similarity in amino acid sequences between carboxypeptidases A and B, as will be discussed below, has strengthened this suggestion and added emphasis to earlier evidence that carboxypeptidases A and B operate by a common mechanism (11, 17, ~$9, 46-47). C. ENDGROUPS AND AMINOACIDSEQUENCES The finding of a one each NH,- and COOH-terminal residue in porcine carboxypeptidase B (Table I) served as evidence that this enzyme is composed of a single polypeptide chain (48).Threonine was identified as the NHz-terminal amino acid by the use of the fluorodinitrobenzene (FDNB) procedure. The NH,-terminal sequence, Thr .Ser, was derived by means of the stepwise phenyl isothiocyanate method. The third residue from the NH,-terminus was tentatively identified by this method as aspartic acid or asparagine. Treatment of the denatured enzyme protein with carboxypeptidase A showed a rapid release of threonine and asparagine followed by a slower release of valine and serine. The hydrazinolysis procedure served to identify threonine as the COOH-terminal amino acid. The NH,-terminal amino acid of bovine carboxypeptidase B, like that of the porcine enzyme, was identified as threonine by the use of the FDNB procedure (40). The suggestion from hydrazinolysis that leucine was the COOH-terminal residue of the bovine enzyme (SO) was confirmed by sequence analysis of a 14-member COOH-terminal peptide fragment derived by cyanogen bromide cleavage of the enzyme protein (Table 111) (49). 43. J. E. Folk and E. W. Schirmer, JBC 238, 3884 (1963). 44. B.L. Vallee and H. Neurath, JBC 217, 253 (1955). 45. J. E. Folk and J. A. Gladner, BBA 48, 139 (1961). 46. J. E. Folk and J. A. Gladner, BBA 33, 570 (1959). 47. T. H. Plummer and W. R. Lawson, JBC 241, 1648 (1966).' 48. J. E.Folk and J. A. Gladner, BBA 47, 595 (1961). 49. M. Elzinga, C. Y. Lai, and C. H. W. Him, ABB 123, 353 (1968).

m

TABLE I11 SEQUENCES OF CERTAIN PEPTIDEFEAGMENTB FBOM BOVINECAEBOXYPEPTIDASE B Fragment

Ref.

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J . E. FOLK

The NH,-terminal residue of the dogfish enzyme has been identified as serine (19). Carboxypeptidase A was found to liberate several amino acids from the denatured enzyme protein. Examination of the rate of release of these residues suggests that leucine occupies the COOH-terminal position (19). Recent studies have revealed sequences of amino acids in several important regions of bovine carboxypeptidase B. These results are given in Table I11 (41, 49-51). An early report on a systematic approach to the complete amino acid sequence of bovine carboxypeptidase B has been made (56). In this case 30 of the possible 36 peptides formed as a result of tryptic digestion of the reduced and aminoethylated enzyme protein were isolated. Of the 300 amino acid residues of carboxypeptidase B, 235 residues were accounted for in these peptides.

D. EABLY CONSIDERATIONS OF HOMOLOGY BETWEEN BOVINE CARBOXYPEPTIDASES A AND B Early interest in comparison of peptide sequences of carboxypeptidases A and B was stimulated by the expectation that these two exopeptidases would show extensive structural homology. This concept, based upon findings of numerous common structural features among enzymes and other proteins of similar biological function (53), would seem valid in light of the similarities between carboxypeptidases A and B in amino acid composition, metal content, enzymic function, and competitive inhibition (11, 17, 40), as well as in parallel consequences of chemical modification (47, 54). Substrate specificity constitutes the primary difference between the enzymes (10, 11, 40). First comparisons of fragmentary peptide segments of bovine carboxypeptidases A and B, however, gave no support to the concept of homology. The amino acid sequences a t the COOH-terminal portion of the two enzymes were found to be distinctly different (49).More puzzling was the finding that the sequences surrounding the single sulfhydryl group of each of the enzyme proteins were clearly not homologous (51). M . Elzinga and C. H. W. Him, ABB 123, 361 (1968). E. Wintersberger, Biochemistry 4, 1533 (1965). M. Elzinga and C. H. W. Him, ABB 123, 343 (1968). V. Bryson and H. J. Vogel, eds., “Evolving Genes and Proteins.” Academic Press, New York, 1965. 54. B. L. Vallee and J. F. Riordan, Brookhaven Symp. Biol. 21, 91 (1968). 50. 51. 52. 53.

2. CARBOXYPEPTIDASE B

67

More recent observations, on the other hand, give renewed interest to the possibility that indeed homology does exist, a t least in important regions of these two enzyme proteivs. The amino acid sequences surrounding an cssential tyrosine residue in each of the enzymes

. -1 Carboxypeptidase B: .1-

Carboxypeptidase A:

Cln -1Ala * Ser Cly

*

Cly * Ser]. Ile

-1

Pro *IAla * Ser * Cly Cly * Serb Asp

(55)

*IAsp.(l4 1 )

have been found to display a remarkable degree of similarity (41). Less convincing comparisons form the basis of an argument for extensive homology in the two bovine enzymes on the levels of both amino acid sequence and three-dimensional structure (56). This case is based upon (a) a postulated zinc-binding position within the disulfide loop of carboxypeptidase B (there are obvious similarities in the amino acid sequences of this disulfide loop and the zinc-binding loop of carboxypeptidase A ) , and (b) a fragment of 14 amino acid residues near the COOH-terminal end of carboxypeptidase A that contains no cysteine residue but displays a sequence similar to that surrounding the sulfhydryl group in carboxypeptidase B. These suggestions present interesting predictions based on the supposition of homology. They seem somewhat premature in support of extensive structural homology, however, when one considers that sequence data for carboxypeptidase B are fragmentary and that no information is available on its three-dimensional structure.

IV. Physical-Chemical Properties and Activation of Procarboxypeptidase B

The catalytically inactive zymogens of carboxypeptidase B from cow (29, 40) and dogfish (19) pancreas gland have been partially characterized, and the mechanism of their conversion to active enzyme has been the subject of preliminary investigations. The sedimentation coefficient, s ~ , , , , ~of, the bovine proenzyme was found to be 4.0S. A molecular weight of 57,400& 1,000 was estimated by short column sedimentation equilibrium with the use of an assumed partial specific volume of 0.73 cm3 g-'. The amino acid composition based on the above molecular weight is as 55. H. Neurath, cited in Plummer (41). 56. R. A. Bradshaw, H. Neurath, and K.A. Walsh, Proc. Natl. Acad. Sci. 406 (1969).

U.S. 63,

68

J. E. FOLK

follows: Asp 47, Thr 37, Ser 44, Glu 47, Pro 19, Gly 34, Ala 33, half-Cys 9, Val 34, Met 10, Ile 22, Leu 36, Tyr 27, Phe 22, Lys 27, His 17, Arg 26, Trp 14, and amide N 46; a total of 505 amino acid residues. No NH,- or COOH-terminal residues have been identified. Dogfish procarboxypeptidase B was found to have an s20,wof 3.67s. A molecular weight in the range of 44,000-45,000 was estimated by means of sedimentation equilibrium analysis. The variations in values reported for apparent weight-average molecular weights and the z-average molecular weights raise some question as to the homogeneity of the isolated zymogen protein. The amino acid composition based on a molecular weight of 44,500 was determined as Asp 46, Thr 28, Ser 27, Glu 35, Pro 17, Gly 26, Ala 31, half-Cys 9, Val 27, Met 8, Ile 24, Leu 25, Tyr 19, Phe 13, Lys 21, His 5, Arg 20, and Trp 13. The NH2-terminal residue was identified as glutamine. Early studies on the activation of procarboxypeptidase B in extracts of fresh bovine pancreas gland and in partially purified preparations of this zymogen showed that trypsin catalyzed the rapid conversion to active enzyme (11). It was also evident from these studies that the activation of the zymogen of carboxypeptidase B occurs much more rapidly than that of carboxypeptidase A. The tryptic activation of purified bovine procarboxypeptidase B has been shown to involve more than a single hydrolytic event (29, 46).A first stage of activation that occurs over a period of 1-2 hr at low trypsin to zymogen molar ratios (1-1000) or within the first few minutes at a 1-10 trypsin to zymogen molar ratio appears to involve hydrolysis of an arginylthreonine bond in the zymogen. I n this stage there is a concomitant (a) appearance of 60-70% of the maximal enzymic activity, (b) appearance of NH2terminal threonine, and (c) release of free arginine, supposedly originating by carboxypeptidase B-catalyzed liberation of a COOH-terminal arginine residue formed as a result of tryptic activation. No change in the sedimentation coefficient occurs during this stage of activation indicating no major fragmentation of the zymogen protein. A second stage of activation that occurs more slowly leads to formation of a protein with a lower sedimentation coefficient and with full enzymic activity. The enzyme formed appears to be identical to crystalline carboxypeptidase B. Major fragmentation of the partially activated protein probably occurs during this stage of activation, e.g., there appears to be a reduction in molecular weight from about 57,500 to 34,000. I n the case of the dogfish proenzyme activation with bovine trypsin results in reduction in molecular weight from 44,500 to 34,000 and change in the NH2-terminal glutamine of the zymogen to NH2-terminal serine of the active enzyme (19).

2.

69

CABBOXYPEPTIDASE B

V. Enzymic Properties of Carboxypeptidare B

A. SPECIFICITY Detailed studies of the substrate specificity of carboxypeptidase B have been carried out with the use of the bovine and the porcine enzymes. Porcine carboxypeptidase B shows little, if any, activity toward carboxypeptidase A substrates (17).The bovine and the dogfish enzymes, on the other hand, have been found to possess a large degree of intrinsic carboxypeptidase A activity (19,29).Examples of these findings are given in Table IV. TABLE IV

ACTIVITIES OF SEVERAL CARBOXYPEPTIDASES TOWARD CARBOXYPEFTIDASE A AND B SUBSTRATE SO.^

Carboxypeptidase Porcine B Bovine Bs Bovine A Dogfish Bh

Carboxypeptidase B substrate hippuryl-carginine (peptide) (uniWmg)

CarboxypeptidaseA substrates

Benzyloxycarbonylglycyl-c mppUryl-D,b phenylalanine (peptide) phenyllactic acid (uniWmg) (eater) (units/mg)

18,400 10,500

Not hydrolysedd

8,350

0.90

Not hydrolyzed!

0.64 3.19'

-

0.22 0.210

0.03

, Units-for hippurylarginine: percent hydrolysis/minute at 1 mill initial substrate concentration; for benzyloxycarbonylglycylphenylalanine:first order rate constant (67) a t 20 mM initial substrate concentration; for hippurylphenyllactic acid: millimolea hydrolyzed/minute a t 10 mM initial substrate concentration. Assay conditions-porcine B: 25 mM tris, 0.1M NaC1, pH 7.65,25"; bovine A and B, dogfish B: for hippurylarginine and hippurylphenyllactic acid, 6 mM sodium barbital, 45 mM NaCl, pH 7.5, 25"; for benzyoxycarbonylglycylphenylalanine, 20 mM sodium barbital, 0.1 M NaC1, pH 7.5, 25". From Folk et al. (17). Reported as no hydrolysis under conditions which would have detected 0.01% of this activity in the carboxypeptidaseB sample (17). From Wintersberger et al. (,%I). Reported as no hydrolysis under conditions where a specific activity of carboxypep tidase A toward this substrate, 0.3% of that of carboxypeptidme B, would have been detected (B9). 0 From Bargetzi et al. (67). From Prahl and Neurath (19). 57. J.-P.Bargetzi, K. 5. V. Sampath Kumar, D. J. Cox, K. A. Walsh, and Neurath, Biochemistry 2, 1468 (1963).

H.

70

J. E. FOLK

1. Peptidase Activity

Early studies on the peptidase activity of bovine carboxypeptidase B were conducted a t a single set concentration of each of the peptide derivatives under test (Table V ) . These simple analyses served to identify certain COOH-terminal basic amino acids as substrates for carboxypeptidase B. More careful later analyses with the porcine enzyme, reported in Section V,B, have defined the relative sensitivity of some of these substrates to carboxypeptidase B-catalyzed hydrolysis. Comparison of the catalytic properties of carboxypeptidase B (Table V) with those of carboxypeptidase A (Chapter 1, this volume) points up the fact that, apart from their specificity toward different COOH-terminal amino acids, these two exopeptidases display striking similarities in their action. These similarities may lx summarized as follows: ( 1 ) They only catalyze the hydrolysis of peptide bonds that are a! to terminal free carboxyl groups; (2) they release only COOH-terminal L-amino acids; (3) they slowly hydrolyze dipeptides having a free amino group, but if this group is acylated the hydrolysis is rapid; (4) the rate at which they release COOH-terminal residues may be greatly influenced by the structure of the penultimate COOH-terminal residue; and ( 5 ) they catalyze the reTABLE V ACTION OF BOVINECARBOXYPEPTIDASE B ON SYNTHETIC PEPTIDE DERIVATIVES' Peptide derivative

% Hydrolysis(min-l mg-l)h

Hippuryl-b-Iysine Hippuryl-D-lysine Hippuryl-clysine amide Hippuryl-cbenzyloxycarbonyl-clysine Hippuryl-targinine Hippurylni tro-carginine Hippuryl-borni thine Hippuryl-6-benzyloxycarbonyl-cornithine Hippuryl-bhomarginine Benzyloxycarbonylglycyl->histidine Glycyl-tlysine c N - ( Benzyloxycarbonylgly cine)-clysine Benzoyl-@-alanyl-b-lysine Hippuryl-cprolyl-b-1 ysine Benzoyl-rAysylgly cine

64.3 0 0

0 46.0 0 19.0 0 0.8 0 C

0 2.2 0.3 0

From Folk and Gladner (11). enzyme as procarboxypeptidase B. Measurements with 0.025 d l peptide derivative in 0.025 M tris buffer, pH 7.65 containing 2 mM diisopropylphosphofluoridate at 25'. c Five to ten percent hydrolysis observed after 5 hr a t 1 mg of enzyme per milliliter. 0

* Milligram

2.

71

CARBOXYPEPTIDASE B

lease of certain amino acids that do not occur naturally but t,hat otherwise satisfy the specificity requirements. It is noteworthy that the COOHterminal basic amino acid, histidine, is not a substrate for carboxypeptidase B. 2. Esterase Activity Carboxypeptidase B was found to catalyze the hydrolysis of hippurylL-argininic acid [o- (hippuryl)-a-L-hydroxy-8-guanidino-n-valericacid] ( 4 6 ) ,the ester analog of hippuryl-L-arginine. That the susceptible peptide bond of a carboxypeptidase B substrate may be replaced by an ester linkage is in accord with the well-known specific esterase activity of the other pancreatic proteolytic enzymes trypsin, chymotrypsin, and carboxypeptidase A. B. KINETICSAND COMPETITIVE INHIBITION Kinetic constants for purified porcine carboxypeptidase B with several peptide substrates and the ester substrate are listed in Table VI. No inhibition by excess substrate was observed with any of the peptide substrates. With the ester, on the other hand, inhibition by excess substrate was observed above the level of 2 mM (32, 46). An interesting feature of the carboxypeptidase B-catalyzed reactions (Table VI) is the pronounced influence of the basic amino acid residue adjacent to the susceptible COOH-terminal residue (benzoyllsyllysine vs. hippuryllysine) and the TABLE VI KINETICCONSTANTS FOR PROCINE CARBOXYPEPTIDASE Bo ~~

Substrate

K, (M x 10')

kob (sec-1)

Peptides Hippuryl-barginine Benzoyl-a-bglu tamyl-barginhe Hippuryl-blysine Benzoyl-blysyl-tl ysine Hippuryl-borthinine

0.21 0.15 7.70 0.18 125.0

105 87 220 86 255

0.04

238

Ester Hippuryl-barginink acid

a From Wolff el al. (38).Assays were conducted a t 23' in 0.025 M tris chloride buffer pH 8.0. * ko(turnovernumber) = moles of substrate hydrolyzed per mole of enzyme per second assuming one active site per molecule of enzyme.

72

J. E. FOLK

lack of influence of the charged carboxyl group of a residue in this adjacent position (benzoylglutamylarginine vs. hippurylarginine) . It is evident from the data of Table VI that, at saturating levels of the hippuryl substrates, ornithine and lysine are released a t approximately the same rates while arginine is liberated at a significantly slower rate. It is likely that the Michaelis constants ( K , values) for these three substrates reflect their relative affinities for the enzyme in the decreasing order arginine, lysine, and ornithine ($2). The insensitive nature of N-acetyl-L-arginine as a substrate [K,, 0.96 x M ; ko,0.02 sec-l (68)] emphasizes the importance of a second peptide bond for rapid hydrolysis. The low K m and KI (Table VII) values for this derivative suggest that the second peptide bond plays a minor role in binding a t the enzyme active site. This is further evident in light of the structural requirements for compounds that display competitive-type inhibition of carboxypeptidase B. A list of these compounds is given in Table VII together with their K , values (inhibitor constants). The inhibition by 8-guanidinovaleric acid and c-aminocaproic acid, together with the lack of inhibition by agmatine and cadaverine, as well as hippuric acid (S2),shows that there are two requirements for binding at the enzyme active site, i.e., a free carboxyl group and the TABLE VII K I VALUESFOR COMPETITIVE-TYPE INHIBITORS OF PORCINE CARBOXYPEPTIDASE Ba Inhibitor

K I (M X 10')

L-Arginine D- Arginine N-Acetyl-barginine N-Acetyl-D-arginine N-Benzoylarginine ~ A r g i n i n i cacid 6-Guanidino-n-valeric acid Wuanidino-n-butyric acid 8-Guanidinopropionic acid Guanidinoacetic acid D,L-Homoarginine Arnithine IrLysine ehinocaproic acid

0.5 0.5 0.8

0.54

0.04 0.25 0.39 4.0 4.0 16.0 1.6 15.0 13.0 1.1

From Wolff et al. ( S d ) . Assay conditions as given in Table VI. The substrate was hippuryl-barginine. 0

68. J. E. Folk, E. C. Wolff, and E. W. Schirmer, JBC 237, 3100 (1962).

73

2. CARBOXYPEPTIDASE B

3.0

2.0 7 -a *O

kE I

I

I

I

7.0

6.0

8.0

9.0

I.o

PH

(a 1

2501

200

I00 6.0

I

9.0 I I 8.0

7.0

PH (b)

FIO.1. Plots of K , and ko against pH for the carboxypeptidase B-catalyeed hydrolysis (a) of the peptide substrate, hippuryl-L-arginine, and (b) of the ester substrate, hippuryl-L-argininic acid. Assays were conducted in 0.025 M trk acetate buffera at 23". From Folk and Wolff (69).

69. J. E. Folk and E. C. WOE, unpublished results (1982).

74

J . E. FOLK

basic side chain. The length of the methylene chain separating the free carboxyl group and the basic group influences the binding properties. The effective inhibition by acetyl-D-arginine which is not a substrate (32) and the fact that hippuryl-D-arginine is neither a substrate nor an inhibitor (32) suggest that there is a spacial arrangement of groupings in the active center of the enzyme that allows ready accommodation of the improperly oriented acetyl group but not the bulky hippuryl moiety (68). The equally effective inhibitory properties of D- and L-arginine are in accord with this suggestion (32) (see also Fig. 2 ) . The effects of pH on the peptidase and esterase activities of carboxypeptidase B is represented in the variation of Kmand ko with pH (Figs. l a and b, respectively) . Variations in binding of several competitivetype inhibitors represented by KI as a function of p H are presented in Fig. 2. Comparison of the recent X-ray solution of the structure of the carboxypeptidase A-substrate complex and model building studies of enzyme-substrate and enzyme-inhibitor interactions (60) (Chapter 1, this volume) with earlier speculations of carboxypeptidase A mechanism, based in part on pH effects on enzymic activity (61, 62), emphasizes the well-recognized pitfalls and limitations of the use of p H effects in assigning ionizable groups as participants in a catalytic mechanism. The obvious similarities between the reactions catalyzed by carboxypeptidases A and B caution against overinterpretation of the pH-activity and pHinhibitor effects of Figs. 1 and 2. Several features of these effects, however, warrant comment. The similarity in form of the curves of K,,, for peptidase activity and of K I for the inhibitor, argininic acid (similar curves were obtained with acetyl-D-arginine and 6-guanidinovaleric acid), suggests that K m reflects substrate binding and that variations in substrate binding over the p H range studied are a function of changes in affinity of enzyme for the free carboxyl group and/or the basic group of substrate. The decrease in ko below pH 7.5 for both peptide and ester substrates suggests participation of the same group of the enzyme in both reactions; the overall forms of the k , curves for these two. reactions, on the other hand, indicate different mechanisms of hydrolysis. The pH-KI curves for D- and Larginine are indicative of inhibition by the species of these amino acids in which the a-amino group is in the unionized form; a plot of -log K I 60. W. N. Lipscomb, J. A. Hartsuch, G . N. Reeke, Jr., F. A. Quiocho, P. H. Bethge, M. L. Ludwig, T. A. Steitz, H. Muirhead, and J. C. Coppola, Brookhaven Symp. Biol. 21, 24 (1968). 61. B. L. Vallee, J. F. Riordan, and J. E. Coleman, Proc. Natl. Acad. Sci. U.S. 49, 109 (1963). 62. F. W. Carson and E. T. Kaiser, JACS 88, 1212 (1966).

2.

75

CABBOXYPEPTIDASE B

\

D-Or L- arginine

6.0

8.0

7.0

9.0

PH

FIG.2. Plots of K Iagainst pH for competitive-type inhibitors of carboxypeptidase B. The substrate was hippuryl-L-arginine.Assay conditions of Fig. 1. From Folk and Wolff (69).

against pH for arginine shows inflections a t p H values identical to those found in this type of plot for both argininic acid and guanidinovaleric acid (69).

C. ACTIVATION AND INHIBITION The inhibition of carboxypeptidase B observed with the metal-chelating agents 1,lO-phenanthroline, 8-hydroxyquinoline-5-sulfonic acid, and 2,2‘dipyridyl is probably a result of binding of the essential metal ion, zinc ( 1 7 ) . The enzyme is inhibited by phosphate and citrate buffers below p H 7.5 and by pyrophosphate and borate above pH 8 ( 3 8 ) .Peptidase activity is not markedly effected by limited changes in ionic strength (NaC1 concentration) (32). l-Butanol and several other alcohols show a pronounced influence on carboxypeptidase B activity (63).Approximately 0.3 M l-butanol causes a 1000/0 increase in the maximum velocity of hippuryl-L-arginine hydrol63.J. E. Folk, E. C. Wolff, E. W. Schirmer, and J. Cornfield, JBC 237, 3105

( 1962).

76

J. E. FOLK

ysis but an 85% decrease in the maximum rate of ester hydrolysis. A kinetic analysis of this phenomenon gives evidence that these catalytic changes are a result of modification of the free enzyme rather than changes in individual rate constants for the catalytic reactions ( 6 3 ) .

VI. Enzymic Properties of Metalloenzymes

Incubation of carboxypeptidase B with salts of cobalt or cadmium a t pH 7.75 results in replacement of the native zinc of the enzyme with 1 g-atom per molecule of the metal used in the incubation ( 4 5 ) . The cobalt and cadmium enzymes display activities toward specific peptide and ester substrates that are markedly altered from those of the native zinc enzyme (Tables VI and VIII). It is apparent from the large variation in turnover numbers for the three metalloenzymes with these substrates that the rates of the overall catalytic reactions are a function of the metal bound to enzyme at the level of 1 g-atom per molecule. I n this regard hippuryl-L-arginine, which is not hydrolyzed by the cadmium enzyme, exerts an effective competitive-type inhibition against the cadM ) (58). mium enzyme-catalyzed esterase activity (&, 0.046 x The differences in KI values for inhibitors of the three metalloenzymes (Tables VII and IX) suggest that the affinity between enzyme and substrate is also influenced by the metal bound to enzyme. There is no reason to suggest, however, that metal ion contributes more than an extrinsic influence in this regard ( 5 8 ) . A unique feature of binding by the cadmium enzyme is the fact that hippuryl-D-arginine, which is neither a substrate nor an inhibitor for the TABLE VIII

KINETICCONSTANTS FOR COBALT AND CADMIUM CARBOXYPEPTIDASES Ba Substrate Peptides Hippuryl-carginine Hippuryl-dysine

Ester Hippuryl-barginink acid

(1

Enzyme

K , (M x 108)

k~ (sec-1)

Cobalt Cadmium Cobalt Cadmium

0.20 220 Not hydrolyzed 5.0 269 Not hydrolyzed

Cobalt Cadmium

0.04 0.36

From Folk et al. (68). Assay conditions as given in Table VI.

119 700

2.

77

CARBOXYPEPTIDASE B

K1

TABLE IX VALUESFOR COMPETITIVE-TYPE INHIBITORS OF COEAWAND CADMIUM Ba CARBOXYPEPTIDASES

Inhibitor ~Argininicacid Acetyl-carginine Acetyl-D-arginine

Enzyme

KI (M x 10’)

Cobalt Cadmium Cobalt Cadmium Cobalt Cadmium

0.91 1.10 1.80 0.78 1.00 0.08

From Folk et al. (68).Assay condition given in Table VI. The substrate was hip puryl-L-argininic acid. 0

zinc and cobalt enzymes and thus is not effectively bound to these enzymes, is bound by the cadmium enzyme (& 0.1 )( M ) (68). VII. Comment on the Enzyme Mechanism

I n consideration of the gross similarities between carboxypeptidases

A and B in terms of molecular size, amino acid composition, metal con-

tent, and enzymic function, as well as the enzymic changes resulting from metal substitution and chemical modification (4‘7, 64) (Chapter 1, this volume), it seems probable that many of the mechanistic features of carboxypeptidase A as determined by X-ray studies (60) (Chapter 1, this volume) will eventually be found common to both exopeptidases. It suffices to say at this point that the several differences in catalytic properties of these enzymes, including the obvious specificity differences, may be expected to be reflected in unique arrangements of chemical groupings in the enzymes’ active sites. VIII. Use in Structural Analysis and Modification of Proteins and Peptides

Methods for the use of carboxypeptidases A and B in structural analyses have recently been outlined in detail (64),as have the purposes and procedures for protein and polypeptide modification by these exo64. R. P. Ambler, “Methods in Enzymology,” Vol. 11, p. 436, 1967.

78

J. E. FOLK

peptidases (66). A few examples of the application of carboxypeptidase

B for the above purposes will serve to acquaint the reader with its

usefulness. Probably the first practical application of carboxypeptidase B to end group determination was in the identification of arginine as the COOHterminal residue of both fibrinopeptides A and B (66) which are released from bovine fibrinogen during its thrombin-catalyzed conversion to fibrin. This finding definitively demonstrated that the proteolytic action of thrombin on fibrinogen is limited to cleavage a t four arginyl peptide bonds per molecule of fibrinogen (MW 360,000). The identification of COOH-terminal lysine in E. coli P-galactosidase (67) presents an example of the use of carboxypeptidase B in protein end group analysis. In this case digestion by carboxypeptidase B was carried out in the presence of the denaturing agent, sodium dodecyl sulfate. The finding of one COOH-terminal lysine per monomer of molecular weight 130,000 was presented as evidence that this enzyme of molecular weight 520,000 is composed of four identical monomer subunits. A novel procedure for identification of the COOH-terminal peptide in tryptic digests of proteins that do not have COOH-terminal basic amino acid residues has been outlined (68). In this method a tryptic digest, applied as a band on paper, is subjected to ionophoresis (pH 6.4). Two strips are cut from the paper parallel to the direction of ionophoresis. One of these is sprayed with a dilute buffered solution of carboxypeptidase B and digestion is allowed to proceed. After the period of digestion the two strips are sewn onto separate sheets of paper and ionophoresis (pH 6.4) is carried out in the second dimension. Because arginine or lysine is removed by carboxypeptidase B from the COOH-terminus of each tryptic peptide except the COOH-terminal one, only the COOH-terminal peptide should appear in the identical position in the digest and the control runs. As an example this method was applied successfully to the y chain of fetal hemoglobin (68). A classic example of protein modification by carboxypeptidases is the selective removal of COOH-terminal residues from the a! and p chains of intact human hemoglobin (69, 70).The COOH-terminal sequences of human hemoglobin are 65. J. T. Potts, Jr., “Methods in Enzymology,” Vol. 11, p. 648, 1967. 66. J. E. Folk, J. A. Gladner, and K. Laki, JBC 234, 67 (1959); J. E. Folk, J. A. Gladner, and Y. Levin, ibid. p. 2317. 67. S. Koorajian and I. Zabin, BBRC 18, 384 (1965). 68. M. A. Naughton and H. Hagopian, Anal. Biochem. 3, 276 (1962).

79

2. CARBOXYPEPTIDASE B .ThrSer-Lys.Tyr-Arg .Ala.His.Lys.Tyr-His

chain fl chain

CY

Carboxypeptidase B removes only arginine from the a chain. Carboxypeptidase A liberates tyrosine and histidine from the /3 chain, while the two enzymes together remove four residues from the /3 chain and at least three residues from the chain (70). These enzymic modifications leave many of the properties of the molecule intact but cause profound and specific changes in the oxygen equilibrium (69, 70). Wide variations were observed in the rates of digestion by both carboxypeptidases A and B of deoxy- and oxyhemoglobin and carboxypeptidase A- or B-modified deoxy- and oxyhemoglobin (70). These findings, together with observed variations in reactivity of the sulfhydryl groups occupying position 93 of the p chains of these forms of hemoglobin, were presented in support of a thesis that irreversible specific conformational changes in the macromolecule are a result of the removal of certain amino acids near the COOH-terminus. A recent report describes the enzymic replacement of the arginyl by a lysyl residue in the reactive site of the soybean trypsin inhibitor (71). Carboxypeptidase B was first employed to remove COOH-terminal arginine from the trypsin-modified inhibtor protein (Arg 64-Ile 65 bond hydrolyzed). By a clever manipulation of reaction conditions, resulting in a reversal of the normal hydrolytic function of carboxypeptidase B, free lysine was inserted into position 64 of the des-Arg-64-modified inhibitor. This was accomplished by the use of a high concentration of carboxypeptidase B and in the presence of free trypsin. The trypsin supplied the driving force for peptide bond synthesis by reacting with the newly synthesized (Lys 64) -modified inhibitor to form trysin-inhibitor complex. Finally, the (Lys 64) inhibitor (Lys 64-Ile 65 bond intact) was obtained by dissociation of the complex with guanidineeHC1. This modified inhibitor protein was found to be comparable in trypsin inhibitory capacity with authentic soybean inhibitor. 1y

69. E. Antonini, J. Wyman, R. Zito, A. Rossi-Fanelli, and A. Caputo, JBC 236, PC60 (1961); M. Brunori, E. Antonini, J. Wyman, R. Zito, J. F. Taylor, and A. Rossi-Fanclli, ibid. 239, 2340 (1964). 70. R. Zito. E. Antonini. and J. Wyman, JBC 239, 1804 (1964). 71. R. W. Sealock and M. Laskowski, Jr., Biochemistry 8, 3703 (1969).