Serine Carboxypeptidases. a Review

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986

SERINE CARBOXYPEPTIDASES. A REVIEW. by KLAUS BREDDAM

Department of Chemistry, Carlsberg Laboratory, Gamle Caflsbergvej 10, DK-2500 Copenhagen Valby

Keywords: Serine carboxypeptidase, acid carboxypeptidase, sequence determination, peptide synthesis, chemical modification, kinetics, site-directed mutagenesis

Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. Those characterized until now can, dependent on their catalytic mechanism, be classified as either metallo carboxypeptidases or as serine carboxypeptidases. Enzymes from the latter group are found in the vacuoles of higher plants and fungi and in the lysosomes of animal cells. Many fungi, in addition, excrete serine carboxypeptidases. Apparently, bacteria do not employ this group of enzymes. Most serine carboxypeptidases presumably participate in the intracellular turnover of proteins and some of them, in addition, release amino acids from extracellular proteins and peptides. However, prolyl carboxypeptidase which cleaves the C-terminal peptide bond of angiotensin II and III is a serine carboxypeptidase with a more specific function. Serine carboxypeptidases are usually glycoproteins with subunit molecular weights of 40,000 - 75,000. Those isolated from fungi apparently contain only a single peptide chain while those isolated from higher plants and animals in most cases contain two peptide chains linked by disulfide bridges. However, a number of the enzymes aggregate forming dimers and oligomers. It is probable that the well-known catalytic mechanism of the serine endopeptidases is also employed by the serine carboxypeptidases but presumably with the difference that the plQ of the catalytically essential histidyl residue is somewhat lower in the carboxypeptidases than in the endopeptidases. However, the leaving group specificity of these two groups of enzymes differ since the carboxypeptidases only release C-terminal amino acids from peptides (peptidase activity) and not longer peptide fragments. In addition, they release C-terminal amino acid amides (peptidyl amino acid amide hydrolase activity) or ammonia (amidase activity) from peptide amides and alcohols from peptide esters (esterase activity) and this property they share with the serine endopeptidases. Like other proteolytic enzymes the serine carboxypeptidases contain binding sites which secure the interaction between enzyme and substrate. In this laboratory, the properties of these have been studied for three serine carboxypeptidases, i.e. carboxypeptidase Y from yeast and malt carboxypeptidases I and II, by means of kinetic studies, chemical modifications of amino acid side-chains located at these binding sites and exchange of such amino acid residues by site-directed mutagenesis. Serine carboxypeptidases, such as carboxypeptidase Y and malt carboxypeptidase II which are available in large quantities, can be applied for several purposes. Their broad specificity and ability to release amino acids from the C-terminus of a peptide chain can be employed in determination of amino acid sequences, and their ability to catalyze transpeptidation reactions and aminolysis ofpeptide esters can be employed to exchange C-terminal amino acid residues in peptides and in step-wise synthesis ofpolypeptides, respectively. The type of reactions catalyzed by these enzymes is limited by their specificities but, fortunately, some of the derivatives of carboxypeptidase Y with changed specificity due to chemical modifications and genetic substitutions of amino acid side-chains located at binding sites can be employed with advantage. These modified enzymes are examples on how the different activities of an enzyme can be perturbed by "protein engineering", hence rendering the enzyme particularly suitable for certain processes.

Springer-Verlag 0105-1938/86/0051/0083/$9.20 K. BREDDAM: Serine carboxypeptidases

Abbreviations: Bu = butyl; Br-Hg-CPD-Y = +Hg-CPD-Y reactivated by bromide; Bz = N-benzoyl; Bzl = benzyl; CPD-Y = carboxypeptidase Y; DFP = diisopropylphosphorofluoridate; FA = furylacryloyl; Hepes = N-2-hydroxy- ethylpiperazine-N-2-ethane sulfonic acid; +Hg-CPD-Y = CPD-Y inactivated by mercuric ions; HPLC = high performance liquid chromatography; I-Hg-CPD-Y = +Hg-CPD-Y reactivated by iodide; Mes = 2-(N-morpho- lino)ethane sulfonic acid; PAB-CPD-Y = CPD-Y modified with phenacylbromide; Ph-Hg + = phenylmercuric ions; Ph-Hg-CPD-Y = CPD-Y modified with phenylmercuric ions; p-HMB = parahydroxymercuribenzoate; Pr = propyl; Z = N-carbobenzoxy. Abbreviations of amino acids, amino acid derivatives and peptides are according to the guidelines of the IUPAC-IUB Commission on Biochemical Nomenclature. The binding site notations for the enzyme is that of SCHECHTER and BERGER (163). Accordingly, the binding site for the C-terminal amino acid residue of the substrate is denoted S; and those for the amino acid residues in the amino-terminal direction away from the scissile bond are denoted S~, Sz,....Si. Amino acid residues in the substrate are referred to as Pj, P~..... P,, and P~ in correspondence with the binding site. A vertical arrow indicates the bond in the substrate being cleaved, henceforth termed the scissile bond.

CONTENTS

Summary ...... 83

Abbreviations ...... 84

1. INTRODUCTION ...... 85

2. DISTRIBUTION AND FUNCTION OF SERINE CARBOXYPEPTIDASES ...... 86 2.1. Serine carboxypeptidases from fungi ...... 86 2.2. Serine carboxypeptidases in higher plants ...... 87 2.3. Mammalian serine carboxypeptidases ...... 88

3. PHYSICO-CHEM1CAL PROPERTIES OF SERINE CARBOXYPEPTIDASES ...... 89

4. THE CATALYTIC MECHANISM OF SERINE ENDOPEPTIDASES ...... 91

5. THE CATALYTIC MECHANISM OF SERINE CARBOXYPEPTIDASES ...... 94 5.1. Modification of catalytically essential amino acid residues ...... 94 5.2. Kinetic evidence for the serine mechanism ...... 95

6. BINDING SITES IN SERINE CARBOXYPEPTIDASES ...... 97 6.1. The influence of the substrate structure ...... 97 6.2. Studies with modifiers ...... 102 6.3. Binding sites for nucleophiles ...... 104 6.4. Chemical modifications of binding sites ...... 106

7. APPLICATIONS OF SERINE CARBOXYPEPTIDASES ...... 112 7. i. Determination of C-terminal sequences ...... 112 7.2. Debittering of bitter peptides ...... 114 7.3. Use of serine carboxypeptidases in peptide synthesis ...... 114 7.4. Exchange of C-terminal amino acid residues in peptides ...... 119

84 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM: Serine carboxypeptidases

1. INTRODUCTION positively charged Arg-145 which binds the It was suggested in 1960 by B.S. HARTLEY(62) C-terminal carboxylate group of the peptide that proteases could be divided into four groups substrate (157), and together with Tyr-248 forms based on the catalytic mechanism employed: l) a dead-end pocket, incapable of accomodating Acid or aspartyl proteases, 2) sulfhydryl protea- more than a single amino acid residue on the ses, 3) serine proteases and 4) metallo proteases. C-terminal side of the scissile bond (153). In This classification primarily comprised endo- contrast, the metallo endopeptidase thermoly- peptidases because the catalytic mechanisms of sin exhibits no restrictions with regard to the exopeptidases were less understood at the time. length of the peptide chain on either side of the However, more recent studies have demon- scissile bond (104). In terms of the nomenclature strated that the bacterial metallo endopeptidase of SCHECHTER and BERGER (163) carboxypep- thermolysin and the mammalian metallo exo- tidase A possesses only a single leaving group peptidase carboxypeptidase A have similar binding site, i.e. S', as opposed to several in constellations of essential amino acid residues in thermolysin, i.e. S;, S~ ..... S?. Three dimensional their active sites (104) and, as shown in the structures of other exopeptidases are not known, present paper, another group of carboxypeptid- but it is highly conceivable that similar dead-end ases exhibit mechanistic similarities with the pockets as observed in carboxypeptidase A ac- serine endopeptidases. Hence, it is reasonable to count for the specificities of all these enzymes. include these two groups ofexopeptidases in the All known carboxypeptidases may be classi- original classification of proteolytic enzymes fied either as metallo carboxypeptidases or as and it is possible that each of the four groups in enzymes belonging to a group which originally the original classification ofproteolytic enzymes by ZUBER and MATILE (204) was termed "acid may in the future be subdivided into endopep- carboxypeptidases" because the optimum for tidases and exopeptidases. their hydrolysis of peptide bonds was in the Exopeptidases of widely different specificities acidic pH range as opposed to the metallo have been isolated: carboxypeptidases and carboxypeptidases which hydrolyze peptides dipeptidyl carboxypeptidases release C-terminal with the highest rates at basic pH values. How- amino acid residues and C-terminal dipeptides, ever, this group of enzymes was renamed by respectively, and analogously aminopeptidases HAYASHI et al. (72) because the original name and dipeptidyl aminopeptidases release N-ter- suggested that they utilized the mechanism of minal amino acid residues and N-terminal the acid proteases and no evidence has been dipeptides, respectively. In addition, a variety of presented for this. On the contrary, all enzymes di- and tripeptidases have been described. It is in this group are inhibited by DFP, a specific characteristic for all of these enzymes that they inhibitor of the serine proteases ( 151 ). The term only cleave peptide bonds at a specific location "serine carboxypeptidases" was therefore relative to the terminus of polypeptide chains in adopted (EC 3.4.16). contrast to endopeptidases which, provided the While the metallo carboxypeptidases are well- side-chain specificity requirements are met, characterized both with respect to phylogenetic have tt~e capacity to cleave a peptide bond relations and catalytic mechanism, much less is regardless of its location in the peptide chain and known about the serine carboxypeptidases and even in the proximity of the terminals. Thus, an only for one of the enzymes, carboxypeptidase Y endopeptidase may also function as an exopep- from yeast, is the primary structure known (36). tidase although normally of low efficiency as However, the interest for these enzymes has demonstrated for trypsin (192). A comparison of increased in recent years as evidenced by the the three-dimensional structures of the active number of publications concerning their cellular sites of carboxypeptidase A and thermolysin location, physiological function, structure, me- illustrates the nature of the restrictions imposed chanism of action, and applications in sequence upon an exopeptidase. The specificity of (met- determination and enzymatic peptide synthesis. allo) carboxypeptidase A towards the C-terminus ofa peptide chain can be attributed to the

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 85 K. BREDDAM: Serine carboxypeptidases

2. DISTRIBUTION AND FUNCTION OF where they appear they presumably serve one of SERINE CARBOXYPEPTIDASES the following purposes: as intracellular enzymes, A literature survey suggests that all carboxy- which are wide-spread in fungi as well as in peptidases found in bacteria are metallo en- higher plants and animals, they participate in the zymes (e.g. 22) since the D,D-carboxypeptidases general turnover of proteins and in providing from various strains of bacteria which contain free amino acids from storage proteins; as extra- an essential serine residue (193, 201) incorrectly cellular enzymes, which primarily are found have been classified as carboxypeptidases: in among fungi, they presumably function to addition to cleaving offa C-terminal amino acid cleave offamino acids needed for nutrition from residue from a peptide these enzymes also cata- external peptides and proteins. lyze transpeptidation reactions with nucleophiles containing several amino acid residues, e.g. pentapeptides (164), and this is in conflict with the nucleophile specificity of the serine 2.1. Serine carboxypeptidases from fungi carboxypeptidases which is limited to single As listed in Table I several strains of penicil- amino acid residues (see section 6.3). Hence, the lum and aspergillus excrete extracellular serine D,D-carboxypeptidases possess several binding carboxypeptidases (87, 202). Aspergillus saitoi sites for the leaving group, i.e. Si, S~..... S~, and in addition contains two intracellular serine cannot be classified as serine carboxypeptidases. carboxypeptidases with immunological proper- As seen from Tables I, II and III the serine ties similar to those of the excreted enzyme, but carboxypeptidases are widely distributed in at present it cannot be established whether these fungi and higher plants, and the observation that enzymes are related or identical (88). lysosomal cathepsin A is a serine carboxypepti- The yeasts Saccharomyces cerevisiae, i.e. dase (47) suggests that these enzymes also are baker's yeast, and Rhodotorula glutinis have common in animal tissues. In all organisms both been shown to contain an intracellular

Table I. Serine carboxypeptidases from fungi Molecular weight Source pI carbo refe- non-disso- dissocia- dissocia- hydrate rence ciating ting, not ting, % conditions reducing reducing

Bakers yeast 64000 n.d. 61000 3.6 15-22 72, 98 Pycnoporus sanguinus 50000 n.d. 54000 4.8 n.d. 89 Aspergillus saitoi 155000 51000 n.d. n.d. n.d. 86 AspergiUus oryzae Type I 120000 n.d. n.d. n.d. n.d 140 Type II 105000 n.d. n.d. n.d. n.d. 141 Type III 61000 n.d. n.d. n.d. n.d. 142 Type IV 43000 n.d. n.d. n.d. n.d. 143 Type 0-1 63000 n.d. n.d. 4.1 n.d. 180, 181 Type 0-2 63000 n.d. n.d. 4.2 n.d. 180, 181 Aspergillus niger 136000 61000 73000 4.1 22 114 Rhodotorula glutinis 80000 n.d. n.d. n.d. n.d. 78 Penicillum janthinellum Type S-! 48000 n.d. 48000 3.7 3 79 Type S-2 135000 65000 n.d. 4.7 7 79 n.d. = not determined

86 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM: Serine carboxypeptidases serine carboxypeptidase (68, 78) while extracel- seggregated into the vacuoles to the same extent lular counterparts have not been identified for as the intact glycoprotein (166). either organism. Carboxypeptidase Y from baker's yeast resides primarily in the vacuoles like proteases A and B and aminopeptidase I 2.2. Serine earboxypeptidases in higher plants (116). Specific inhibitors of each of these en- Serine carboxypeptidases have been found in zymes are located in the cytoplasm surrounding various fruits and seeds (Table II) (49, 50, 109, the vacuoles (16, 39, 118, 119, 125, 146, 161) 110, 111, 127, 128, 165, 188, 205) and some- presumably regulating proteolysis (39). times they are induced during germination (49, Carboxypeptidase Y is synthesized as a 59,000 52, 91,92, 152). Plant stems and leaves have also molecular weight precursor (139) which, due to been shown to contain serine carboxypeptidases glycosylation and incorporation of phosphate, (41, 49, 171, 191) and in one of these cases attains a molecular weight of 67,000 in the wounding of a single leaf caused a three fold endoplasmatic reticulum and subsequently, one increase of carboxypeptidase activity in other of 69,000 in the Golgi body (175). This inactive unwounded leaves ( 191 ). precursor is converted to the active enzyme by ZUBER and MATILE demonstrated in 1968 cleavage of a peptide bond near the N-terminus (204) that the serine carboxypeptidases in higher of the zymogen thereby releasing a peptide with plants were intracellular enzymes localized in a molecular weight of 6,000 - 9,000 (45, 65, 75). vacuoles and they assumed that they functioned However, the proteolytic enzyme involved in in intracellular protein turnover. This obser- this activation and its location in the cell is vation has been confirmed in the more recent unknown, but it is probable that this process literature, but the picture is complex since higher takes place subsequent to transferral of the plants are composed of many organs and tissues. precursor from the Golgi body to the vacuole Thus, in rice plants three apparently different (175). Other enzymes from yeast vacuoles (134) serine carboxypeptidases have been found (49, and from the lysosome (66, 67, 170), the orga- 50). One was found in the leaves and it also nelle in animal cells with equivalent function appeared in the seeds during germination. An- (126), are similarly synthesized as precursors. other enzyme was primarily present in resting This principle of protein synthesis may serve seeds and it decreased during germination, and several purposes to cells: it may help the correct a third enzyme was transiently present in young folding and crosslinking of peptide chains dur- roots and shoots. These results suggest that ing protein synthesis, e.g. proinsulin (172), or in different tissues of plants have different carboxy- cases where the precursor is inactive it may peptidases in amounts which depend on the protect the cell against unwanted enzymatic developmental stage of each tissue. However, it action, e.g. trypsinogen (144). Alternatively, an has not been established whether these carboxy- additional N-terminal peptide segment may peptidases represent isoenzymes. The carboxy- function as a signal peptide guiding the enzyme peptidases isolated from germinated barley to the correct location in the cell (75) in accord- grains exhibit a similar complexity (11, 28, 37, ance with the hypothesis of BLOBEL and co- 135, 154, 190, 200). workers (18, 40). Studies by T. STEVENS et al. The serine carboxypeptidase induced during (176) have established that a signal sequence of germination of cotton seeds functions together 15 amino acid residues near the N-terminus is with other proteolytic enzymes to release amino responsible for the translocation ofpro-carboxy- acids from storage proteins and thus support the peptidase Y across the endoplasmatic reticulum growth of the emerging tissues of the seedling and that another short sequence is required for (91, 92). The synthesis of this carboxypeptidase efficient delivery of the protein to the vacuole. stops when the rate of disappearance of the The carbohydrate portion of carboxypeptidase storage protein is maximal. The carboxypeptid- Y (see section 3) serves no such recognition ases from germinated barley have similar func- functions since the carbohydrate-flee enzyme tions, rendering these enzymes important in the synthesized in the presence oftunicamycin was production of beer (52). In this process carboxy-

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 87 K. BREDDAM: Serine carboxypeptidases

Table I1. Serine carboxypeptidases from higher plants

Source Molecular weight pI carbo refe- non-disso- dissocia- dissocia- hydrate rence ciating ting, not ting, % conditions reducing reducing Barley (malt) 19000 Carboxypeptidase I 100000 51000 32000 5.7 8 28

27000 Carboxypeptidase II 120000 61000 34000 n.d. 15 37

21000 Rice bran 110000 n.d. 7.8 n.d. 50 35000 25000 Wheat bran 118000 58000 6.0 12 188 35000 18000 Leaves of wounded 105000 n.d. 5.2 n.d 191 37000 tomato plants 24000 Germinatingcotton 85000 n.d. 31000 n.d. 1 91 seedings 33000 Germinated wheat Type A 63000 n.d. 60000 5.7. n.d. 152 Type B 54000 n.d. 56000 n.d. n.d. 152 Rice leaves 120000 n.d. n.d. 6.0 n.d. 49 Watermelon Type F-I 89000 n.d. n.d. 4.4 n.d 127 Type F-II 110000 n.d. n.d. 5.0 n.d 127 Citrus Natsudaidai 93000 n.d. n.d. n.d 10 109 Citrus orange 120-150000 n.d. n.d. n.d. n.d. 205 Orange leaves 175000 n.d. n.d. 4.5 n.d. 171 Mandarin orange Type C~a 96000 n.d. n.d. 4.8 7 57 Type Cub 112000 n.d. n.d. 4.7 6 57 n.d. = not determined

peptidases are responsible for the degradation of 2.3. Mammalian serine carboxypeptidases polypeptides released from the kernels by endo- Among the intracellular lysosomal proteoly- peptidases. After mashing, I0-15% of the grain- tic enzymes, cathepsin A (47, 48, I00, 101,102, protein has been converted to free amino acids 130) has been positively identified as a serine (52). In this context it has also been suggested carboxypeptidase. The catalytic properties of that a chelator-insensitive carboxypeptidase the enzyme named cathepsin B2 suggest that it during malting solubilizes 13-glucans released also is a carboxypeptidase (1) but inhibition data from the barley, presumably by releasing amino have so far not been presented. Dol (47) has acids from the glucan (9). argued that catheptic carboxypeptidase and ca-

88 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM"Serine carboxypeptidases

Table III. Serine carboxypeptidases from mammalians Molecular weight Source pl carbo refe- non-disso- dissocia- dissocia- hydrate rence ciating ring, not ring, % conditions reducing reducing Rat liver cathepsin Type Af 100000 n.d. several 4.7 n.d. 130 Type AH 200000 n.d. several 4.8 n.d. 130 Type A.[ 420000 n.d. several 4.9 n.d. 130

Pig kidney cathepsin 2000O Type A,L 500000 n.d. 25000 5.8 n.d. 100, 102 50000 20000 Type A,S 100000 n.d. 25000 5.0 n.d. l01,102 50000 66000 Human kidney prolyl 115000 n.d. n.d. n.d. 147 45000 carboxypeptidase n.d. = not determined

thepsin A is really the same enzyme, but both tidases, with the exception of the one from rice enzymes are incompletely characterized with leaves, are acidic proteins which, apart from the respect to physico-chemical and immunological one from cotton seeds, contain covalently linked properties. The function of the lysosomal en- carbohydrate. The molecular weights range zymes in mammals is presumably similar to the from 43,000 to approximately 500,000 under function of the enzymes localized in the vacuol- non-dissociating conditions, i.e. as determined es of yeast cells. by gelfiltration or ultracentrifugation in the The enzymes in various human organs, in absence of urea, SDS etc. Where measurements urine and in leucocytes which cleave the C-ter- were performed under dissociating conditions minal Pro-Phe peptide bond in angiotensins II but in the absence of reducing agents, e.g. and III - thereby inactivating their hypertensive mercaptoethanol, molecular weights of 45,000 activity - have also been demonstrated to be to 65,000 were obtained, suggesting that the serine carboxypeptidases (147, 148). The en- higher molecular weights are due to association zyme isolated from the kidney is presumably of monomers into dimers or oligomers. located in the lysosome, but its importance in ICHISHIMA (86) has demonstrated for the carbo- regulation of the level ofangiotensin and hence xypeptidase from Aspergillus saitoi that the blood pressure is still unknown (147). association is influenced by the salt concentration: in the presence of NaCI the molecular 3. PHYSICO-CHEMICAL PROPERTIES weight is around 140,000, and in the absence of OF SERINE CARBOXYPEPTIDASES NaC1 the enzyme exists in two forms of molec- Tables I, II, and III list the molecular weights, ular weights 51,000 and 140,000, respectively. isoelectfic points and contents of carbohydrate However, similar studies of the influence of ofserine carboxypeptidases isolated from fungi, ionic strength on the association properties have higher plants and animals, respectively. It is not been performed with other serine carboxy- observed that all investigated serine carboxypep- peptidases.

Cadsberg Res. Commun. Vol. 51, p. 83-128, 1986 89 K. BREDDAM"Serine carboxypeptidases

With respect to the number of peptide chains bridges, such that the active species comprises in the molecule important differences are appar- two peptide chains linked by disulfide bridges ent between enzymes isolated from fungi (Table (145) and this could similarly account for the I) and those isolated from higher plants (Table existence of two peptide chains in the serine II): serine carboxypeptidases from fungi contain carboxypeptidases from plants. The existence of only a single peptide chain with a molecular only a single peptide chain in the active enzymes weight of 48,000 - 73,000 (including attached from fungi suggests that potential zymogens of carbohydrate) while those isolated from tomato these enzymes might be activated by cleavage of plants, rice bran, wheat bran and germinated an internal peptide bond located on the N-ter- barley all contain two peptide chains of mol- minal side of the disulfide bridges. This mech- ecular weights 32,000 - 37,000 for the largest anism of activation has been demonstrated for chain and 19,000- 27,000 for the smallest chain. the zymogen ofcarboxypeptidase Y (see section Since these enzymes under non-dissociating 2.1). conditions are characterized by molecular The amino acid composition of several serine weights of 100,000 - 120,000 it may be assumed carboxypeptidases has been determined (28, 37, that they normally are dimers and that the 79, 91, 98, 110, 111, 114, 188, 191) and it is monomers of molecular weights 50,000 - characteristic that the enzymes isolated from 60,000 are composed of two different peptide fungi have much higher contents of the amino chains linked by disulfide bridges. This structure acids Asx and Glx and lower contents of the may well be shared by other serine carboxypep- basic amino acids, i.e. Lys and Arg, than those tidases isolated from higher plants which have from higher plants. All the enzymes contain not been investigated in similar detail. The disulfide bridges, and the enzymes from yeast (6, enzyme from germinating cotton seedlings was 27) penicillum (79) and carboxypeptidase I from shown to contain three peptide chains of mol- barley (28) have been shown in addition to ecular weights 33,000, 31,000 and 24,000, but it contain a single buried sulfhydryl group (further is conceivable, as pointed out by the authors, details are given in section 6). Approximately that the peptide chain of molecular weight 98% of the amino acid sequence ofcarboxypep- 31,000 is derived from the one of molecular tidase Y has been identified by SVENDSEN and weight 33,000, such that this enzyme in reality is coworkers (124, 179) utilizing traditional Ed- composed of two peptide chains. man degradation, and DNA sequencing has The limited data available on the subunit recently completed the primary structure (T. structure of serine carboxypeptidases from STEVENS, personal communications) (36, 176). animals (Table III) suggest similarities with the At present, approximately 80% of the amino structure of the serine carboxypeptidases from acid sequences of the A-chains of malt carboxy- higher plants. The cathepsins A,S and A,L from peptidases I and II are known and they indicate porcine kidney have molecular weights of 35% homology with each other and approxi- 100,000 and 500,000, respectively. However, mately 30% homology with the N-terminal por- both enzymes are composed ofpeptide chains of tion of yeast carboxypeptidase (S. SORENSEN, K. molecular weights 20,000, 25,000 and 55,000 BREDDAM, I. SVENDSEN and M. OTTESEN, un- (102), which might indicate that the two en- published observations) suggesting that the se- zymes contain identical subunits but different rine carboxypeptidases from higher plants and degrees of association. those from fungi are related. Very little homo- The structural differences between serine car- logy with sequences of other known proteolytic boxypeptidases from fungi and those from enzymes has been observed, suggesting that higher plants and animals could be due to serine carboxypeptidases are not or only different mechanisms of converting the zymo- distantly related to other serine proteases. gens to active enzymes. Several zymogens, e.g. Apart from the enzyme from cotton seedlings chymotrypsinogen, have been shown to be acti- (91) all the serine carboxypeptidases, which have vated by a proteolytic cleavage of internal pep- been analyzed for carbohydrate content, have tide bonds located between two disulfide turned out to be glycoproteins (Table I) contain-

90 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM:Serine carboxypeptidases ing neutral sugar (galactose and/or mannose) as explains why an apparently homogeneous prep- well as glucosamine. Carboxypeptidase Y con- aration of yeast carboxypeptidase (98) with no tains four carbohydrate moieties which are at- evidence for the existence of isoenzymes as tached to the protein through asparagine side evaluated from sequence studies (179) has been chains (65, 183) at positions tentatively identi- shown to contain at least two species with fied by amino acid sequencing (36). All four different isoelectric points and slightly different positions are characterized by an Asn-X-Thr/ activities (98). It has not been clarified whether Ser sequence which is typical in glycoproteins the phosphate and/or carbohydrate attached to with N-linked carbohydrates (85). Like in other the enzyme influence enzymatic activity. En- enzymes from the vacuoles of yeast and from the zymatic removal of the three accessible lysosomes of mammalian cells (64, 66, 67, 184) carbohydrate moieties did not alter the peptid- each carbohydrate moiety in carboxypeptidase ase activity of carboxypeptidase Y (183), but Y is composed of two N-acetylglucosamine resi- since denaturation of the enzyme was needed to dues and a branched mannose moiety (63, 184). remove the fourth moiety, this could not be fully Three of these carbohydrate moieties may be answered (43, 183). More direct evidence might cleaved off by endo-13-N-acetylglucosaminidase be obtained by kinetic characterization of the H and each of these contains from 11 to 18 carbohydrate-free carboxypeptidase Y pro- mannose residues and I or 2 phosphate residues. duced by yeast grown in the presence oftunica- The fourth moiety which is only released from mycin (65) but such results have hitherto not denatured carboxypeptidase (183) contains 8 - been described. It may be noted that malt 12 mannose residues and no phosphate (184). carboxypeptidase I also exists in two species with This moiety is presumable attached before final identical N-terminal sequences but different folding of the protein with the consequence that isoelectric points. The reasons for this hetero- it becomes inaccessible rendering further attach- geneity may be the same as for yeast carboxypep- ment of mannose residues impossible as op- tidase. posed to the three accessible carbohydrate moieties (184). The heterogeneity of the carbohydrate portion of carboxypeptidase Y has resulted in en- 4. THE CATALYTIC MECHANISM OF zyme preparations of widely different carbohy- THE SERINE ENDOPEPTIDASES drate content (120) and enzyme with four The results of chemical modifications and phosphate groups has been separated from en- kinetic studies during the 1950ies and 60ies zyme with five phosphate groups by ion ex- suggested that a seryl and a histidyl residue were change chromatography (63). This probably involved in the catalytic mechanism of the serine

Acylation

CH2 I ,o . ,o HN ~N H I0 H~_INH R-C-XI --"-7 HN,,,,,,,~,N C=O| + R-C-X O- +HX R II O Deacylation Scheme 1. The reaction mechanism ofthe serine proteases as proposed by BENDERand KEZDY(12). Im = imidazole, X = OR or NHR in the acylation and OH in the deacylation reaction.

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 91 K. BREDDAM: Serine carboxypeptidases endopeptidases, and based on these obser- from the seryl -OH to the leaving group in the vations BENDER and KEZDY (12) proposed the acylation reaction. After years of dispute over reaction mechanism shown in Scheme I. It is the state of ionization of the His-Asp couple seen that the nucleophilic attack by the hydroxyl ROBERTS and coworkers (4, 5) have now con- group of the reactive seryl residue on the carbo- vincingly demonstrated, by means of'SN and '3C nyl carbon atom of the substrate is catalyzed by NMR spectroscopy that the pK~ of the imidazo- a histidyl residue functioning as a general base. lium cation of the histidyl residue of ct-lytic This leads to the formation of a tetrahedral protease is approximately 7, and that the en- intermediate and an imidazolium ion. The in- zyme is in its active form, i.e. pH > 7, when the termediate breaks down by general acid catalysis histidyl side-chain is protonated at N-3 and to an acyl-enzyme, an imidazole base, and an unprotonated at N-1. They consequently sug- alcohol or amine depending on the nature of the gested that the tetrahedral adduct is formed by substrate. The acyl-enzyme is hydrolysed the mechanism shown in Scheme 2 which through the reverse reaction pathway with water deviates from the mechanisms proposed by functioning as nucleophile instead of the hydro- other investigators (19, 84) by the aspartyl side- xyl group of the seryl residue. The first X-ray chain remaining unprotonated during the reac- crystallographic studies of chymotrypsin con- tion. It was suggested that this negatively firmed that a histidyl residue is located in the charged group instead functions in one of the proximity of the active site seryl residue (131) following ways: a) to favour the correct imida- and later crystallographic studies have provided zole tautomer, b) to orient the imidazole ring, or support for the existence of both tetrahedral (83) c) to stabilize the imidazolium cation. All these and acyl-enzyme intermediates (76, 77) in the studies were performed with "resting" enzyme, reaction sequences of these enzymes. i.e. in the absence of substrate, but crystallo- X-ray crystallography has furthermore re- graphic studies on the complex between trypsin vealed that the histidyl side-chain also interacts and pancreatic trypsin inhibitor have suggested with a buried aspartyl side-chain (19) and it is that a hydrogen bond between the seryl and generally accepted that all three amino acid histidyl residues does exist (83) and NMR stu- residues, i.e. Ser, His, and Asp, are functionally dies of this enzyme-inhibitor complex imply essential because they are present in all serine partial formation of an alkoxide anion on the endopeptidases which so far have been investi- seryi residue (121). These results are in conflict gated by X-ray crystallography. BLOW et at. (19) with those obtained by KRAUT and coworkers proposed that the side-chains of Asp, His, and ( 108, 132), suggesting that important differences Ser form a triad linked by hydrogen bonds exist between "resting" and "working" enzyme thereby constituting a "charge relay system" (174), such that observations with resting en- which functions by a partial transfer of the zyme are insufficient to explain important hydrogen atom of the hydroxyl group of the seryl aspects of its function. residue to the aspartyl carboxylate group, hence X-ray crystallography studies on several se- forming a strongly nucleophilic alkoxide group. fine endopeptidases suggest that the binding However, KRAUT and coworkers (108, 133) sites responsible for the proper interaction be- have shown for several serine endopeptidases- tween enzyme and substrate are of three catego- that such a hydrogen bond does not exist be- ries: (a) binding sites for the backbone of poly- tween the seryl and the histidyl residues and peptide substrates, (b) binding sites for the consequently, the seryl side-chain could hardly amino acid side-chains, and (c) the oxyanion be strongly nucleophilic. They proposed that the binding site. seryl residue reacts with the substrate only The interaction between enzyme and the because it is in the optimum position to attack backbone of the acyl portion of polypeptide the substrate's tetrahedrally distorted carbon substrates was first observed in chymotrypsin atom which has been induced by its binding to (167) and in subtilisin (107, 158). However, the enzyme. Thus, the only function of the similar interactions with the backbone of the His-Asp couple would be to transfer a proton imine portion of peptide substrates have not

92 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM: Serine carboxypeptidases

o _ tJ~',,.,u_fi HN N: -H I " -'6' ~"'n2 v" 0 - V R o- ,-6-x

Scheme 2. Mechanism of formation of the tetrahedrat intermediate as proposed by BACHOVCHINand ROBERTS(4).

been demonstrated. In subtilisin X-ray crystallo- termine their specificity but they probably also graphy has indicated the existence of four bind- function to stabilize the transition state configu- ing sites for the side-chains at the P,, P2, P3, and ration. ROBERTUS et al. (159) have suggested P4 positions of the substrate (158, 159), and for that this is partially achieved by an increase in trypsin, chymotrypsin, and elastase similar re- the binding strength between enzyme and sub- suits have been obtained (169, 173, 177). A strate at the P, position as the Michaelis complex crevice in chymotrypsin for the side-chain in the is converted into the tetrahedral intermediate P, position is hydrophobic and shaped such that and HENDERSON (76, 77) has proposed the aromatic side-chains fit much better than other existence of a specific binding site for the oxyan- hydrophobic and hydrophilic side-chains (173). ion in the serine endopeptidases. He suggested A corresponding cleft in trypsin is similar in that hydrogen bonds were formed between the geometry, but it contains a negatively charged substrate carbonyl oxygen of the acyl-enzyme aspartyl side-chain which renders the enzyme intermediate and two amide groups in the back- specific for substrates with a positively charged bone of chymotrypsin, and that these bonds side-chain, i.e. Lys and Arg, in the P, position might be stronger in a tetrahedral transition (177). In elastase two bulky side-chains protrude state. A true tetrahedral transition state with a into the hydrophobic crevice for the amino acid covalent bond between the serine at the active side-chain at the P, position hence, reducing its site of the enzyme and the substrate has not yet size and consequently, elastase allows the bind- been observed, but X-ray crystallography of the ing of the methyl side-chain of an alanyl residue complexes between trypsin and pancreatic tryp- in the substrate much better than more bulky sin inhibitor indicate that the reactive carbonyl side-chains (169). Subtilisin is characterized by a group in the inhibitor is tetrahedrally distorted much wider specificity than chymotrypsin, tryp- with the oxygen atom hydrogen bonded to two sin, and elastase and consistent with this it amide groups of the backbone of the enzyme contains a binding site for the side-chain at the P~ located in the oxyanion binding site (83). How- position which is sterically much less restrictive ever, it appears that no covalent bond has been than in the other enzymes. X-ray studies of the formed between the substrate and the seryl complexes between trypsin and pancreatic tryp- residue in the active site since an identical sin inhibitor (83) have indicated that the side complex is formed with trypsin where the active chains of the amino acid residues in the P] and P'_, site serine has been converted to dehydroalanine positions of both inhibitors interact with the (83). Hence, the intermediate may be considered enzyme but in a less specific manner than a pretransition state Michaelis complex as op- observed on the other side of the scissile bond. posed to a true transition state, and its formation However, kinetic studies of several serine endo- is apparently dependent on the existence of a peptidases have provided evidence for the im- complementary surface on the enzyme, i.e. the portance of interactions between enzyme and oxyanion binding site and the binding sites for the side-chains on both sides of the scissile bond the side-chains and backbone of the substrate. as reviewed by SVENDSEN (178). However, the results are controversial since The binding sites of proteolytic enzymes de- NMR studies have failed to provide confirma-

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 93 K. BREDDAM: Serine carboxypeptidases tion of the tetrahedrally skewed carbonyl group Enzymes Amino acid sequences (155). Carboxypeptidase Cua -Glu-Gly-Asp-Ser-Gly-Gly-Glu-Leu~ Additional evidence for the existence of an oxyanion binding site has been obtained by Cacboxypeptidase CUb -Glu-Gly-Asp-Ser-G]y-Gly-G1 u-Leu- studying the three dimensional structures of Cacboxypeptidase Y -Ala-Gly-Glu-Ser-Tyr-Ala-His-Gly- complexes between serine endopeptidases and Trypsin(bovine) -Gln-Gly-Asp-Ser-GIy-Gly-Pro-Val- transition state analogues, i.e. substances which Chymotrypsin(bovine) -Met-Gly-Asp-Ser-G]y-G1y-Pro-Leu- with the reactive seryl residue form stable ad- Elastase(pig) -Gln-Gly-Asp-Ser-Gly-Gly-Pro-Leu- ducts with tetrahedral structures resembling the Plasmin(human) -G1n-Gly-Asp-Ser-G1y-Gly-Pro-Leu- proposed transition state shown in Scheme 1. Thrombin(bovine) Thus, in trypsin treated with DFP an oxygen -G1u-Gly-Asp-Ser-Gly-G1y-Pro-Phe- atom of the diisopropylphosphoryl group ac- Subtilisin BPN -Asn-Gly-rhr-Ser-Met-Ala-Ser-Pro- cepts hydrogen bonds from the two amide Scheme 3. Amino acid sequences around the reactive groups located in the proposed oxyanion bind- seryl residue (marked with an asterisk) of typical serine ing site (177). Phenylboronic acid, 2-phenyl- proteases. Data from ref. 57. ethylboronic acid ( 105, 117, 132), phenylarsonic acid (17, 59), and various aromatic sulfonyl flourides (76) have been shown to form similar tetrahedral adducts to the active site serine tion has been identified as a seryl residue in three hydroxyl group in serine endopeptidases. of these enzymes, i.e. carboxypeptidase Y (71) and carboxypeptidasesCua and Cub from mandarin orange (57). The amino acid sequence 5. THE CATALYTIC MECHANISM OF around the reactive seryl residue is compared SERINE CARBOXYPEPTIDASES with those of subtilisin and mammalian serine As discussed in section 4 two different features endopeptidases in Scheme 3. It is observed that are responsible for the catalysis of serine endo- similarities exist between the carboxypeptidases peptidases: a) a catalytic triad, consisting of Ser, from mandarin orange and mammalian endo- His, and Asp, and b) multiple binding sites for peptidases while the sequence around the seryl the substrate. Similar features are probably re- residue in carboxypeptidase Y exhibits only sponsible for the catalytic activity of serine slight analogy with other enzymes. It should be carboxypeptidases, but short of X-ray studies an noted that all the listed enzymes contain a glycyl evaluation of their catalytic properties must be residue in position 3 in the N-terminal direction based on the results of kinetic studies, chemical of the active site seryl residue suggesting it to modifications, and site-directed mutagenesis. have arisen as a result of a convergent evolution. Various chloromethylketones have been demonstrated to specifically modify the essen- 5.1. Modification of catalytically essential tial histidyl residue of the serine endopeptidases amino acid residues ( 150, 168), and HAYASHIet al. (69) have demon- DFP has been shown to specifically modify strated that a reagent of this type also modifies a the seryl residue in the active site of numerous single histidyl residue in carboxypeptidase Y serine endopeptidases causing inactivation of abolishing both esterase and peptidase activities. these enzymes (e.g. 151). In fact, inactivation of Unfortunately, the position of the modified a proteolytic enzyme with this reagent has be- residue in the sequence has not yet been identi- come presumptive evidence for the involvement fied. The involvement ofa histidyl residue in the ofa seryl residue in the catalytic mechanism. All catalytic mechanism of other serine carboxy- activities of the carboxypeptidases listed in Ta- peptidases has similarly been implicated: the bles I to III are inhibited by DFP demonstrating carboxypeptidase from Aspergillus saitoi is also that a seryl residue is essential for their catalytic inhibited by a chloromethylketone (86), and the activity, hence justifying their classification as carboxypeptidases from Penicillum janthinel- serine carboxypeptidases. The site of modifica- lure (79) and mandarin orange (112) are inhi-

94 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM; Serine carboxypeptidases bited by photooxidation. The probable importance ofa seryl and a histidyl residue suggest that 5000 I 0.5 serine carboxypeptidases employ a mechanism similar to the one employed by the serine endopeptidases. This is supported by the fact that carboxypeptidase Y, as shown in this laboratory, is inhibited by p-aminophenylarsonic acid and 2500 ).25 phenylboronic acid (98), two compounds which are considered to be transition state analogues ._ for the serine endopeptidases (see section 4). E n Q o o However, as yet no evidence has been presented I I i i J i I= for the involvement of an aspartyl residue in the El o catalytic mechanism of the serine carboxypep- IOOOO y E tidases. 7

3 5.2. Kinetic evidence for the serine mechanism In the reaction sequence of carboxypeptidase 5000 2 Y catalyzed hydrolysis reactions only indirect evidence points to the existence of an acyl-en- 1 zyme intermediate (122). DOUGLAS et al. (51) have by means of stopped-flow spectrophoto- I I merry observed burst kinetics in the carboxypep- 4- 5 6 7 8 9 tidase Y catalyzed hydrolysis of 4-nitrophenyl pH trimethylacetate and shown this to be in agree- Figure 1. The influence of pH on I~, and Km for ment with the existence ofa trimethylacetyl-en- carbox{peptidase Y catalyzed hydrolysis of the ester zyme in the reaction course. However, for a Bz-Ala-OBzl (Panel A) (27) and the peptide FA-Phe-~ number of the serine carboxypeptidases listed in Ala-OH (panel B) (34). Tables I, II and III it is observed that esterase activity is optimal approximately two pH units higher than peptidase activity and this differs from the serine endopeptidases for which the ionizable group with a pKa below 5 and remains hydrolysis of all substrates is dependent on the constant in the pH range 6 - 9.5. Similar results deprotonation ofa histidyl residue (4) with a pK~ have been reported by HAYASHI using Ac-Phe around 7.0 (12). This brings up the question OEt as substrate (70). For the peptide substrates whether the influence of pH on the various Z-Phe (70) and FA-Phe (34) activities of the serine carboxypeptidases is com- k~a, is dependent on the deprotonation of a group patible with the proposed mechanism of the with a pKa of 5.4 while Km increases with serine endopeptidases. protonation of a group with a pKa around 7 Among the serine carboxypeptidases the in- (Figure 1B). Thus, the different pH optima for fluence of pH on the kinetic parameters of ester hydrolysis of ester and peptide substrates are and peptide hydrolysis have only been studied primarily due to the influence of pH on Km for for carboxypeptidase Y and malt carboxypep- the hydrolysis of peptide substrates. For amide tidase II. With the former enzyme the present substrates the pH dependence ofk~ar and Km have author has shown (27) that k,, for the hydrolysis not been determined because these substrates of Bz-AIa is dependent on the deprotona- cannot be dissolved in sufficiently high concen- tion of an ionizable group with a pI~ of 5.2 - 5.5 trations, but the pH profiles for hydrolysis rates and it remains constant in the pH range 7.5 - 9.5 suggest similarities with ester substrates (8, 28), (Figure 1A). Km for the hydrolysis of Bz-Ala presumably due to the fact that ester and amide OBzl is dependent on the deprotonation of an substrates both contain a blocked C-terminal

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 95 K. BREDDAM"Serine carboxypeptidases

Ks k2 k3 Assuming the reaction sequence of the serine E + S e . ES - ) ES' > E + P2 + endopeptidases (Scheme 4) to be valid for the serine carboxypeptidases as well, it is possible to P1 estimate the relative magnitudes of k2 and k3 for some of these enzymes. For carboxypeptidase Y k2-k 3 k3 (70), watermelon carboxypeptidase (129) and kcat =k-~ Km = Ks " k-~ cathepsin A (103) it has been demonstrated that k~a, for the hydrolysis of N-blocked dipeptides is Scheme 4. Reaction mechanism for the serine protea- strongly dependent on the nature of the leaving ses. E = enzyme; S = substrate; ES = Michaelis group, i.e. the C-terminal amino acid residue. A complex; K~ = dissociation constant ofthe ES complex; similar dependence ofk~, on the alcohol leaving ES' = the acyl-enzyme intermediate; P~ = leaving group of ester substrates has been demonstrated group; P2 = hydrolysis product; k2 and k3 are the rate for carboxypeptidase Y (8, 27), malt carboxy- constants for the acylation and deacylation reactions, respectively. peptidases I and II (30, 38). With the serine endopeptidases the leaving group influences k~a, for the hydrolysis of amide bonds while for the hydrolysis of ester substrates it is independent of carboxylic acid group. Similar observations the nature of the leaving group. This is consistent have been made with malt carboxypeptidase II with k2 ~ k~ for the hydrolysis of amide bonds but with the difference that K~t for the hydrolysis and k2 ~> k3 for the hydrolysis of ester bonds (12), of ester and peptide substrates, i.e. FA-Ala-~OBzl and consequently, with the serine carboxypep- and FA-Phe-*Phe-OH, respectively, is dependent tidases k2 ~ k~ for the hydrolysis of both amide on the deprotonation of a group with a pig and ester bonds. However, the limited data must around 4.0 (38). be evaluated with caution since the situation The fact that k~, for the hydrolysis ofboth ester where k2,~ k3 cannot be distinguished from k2 and peptide substrates depend on ionizable k3 as long as only the steady-state kinetics of groups with pKa values around 5.4 and 4.0 for hydrolysis reactions have been studied. Deter- carboxypeptidase Y and malt carboxypeptidase mination of kinetic parameters in the presence II, respectively, suggest that the ionization of a of added nucleophiles is more informative and single residue in both enzymes determine their such studies have been performed with malt activities towards all substrates regardless of carboxypeptidase I in this laboratory using Z- whether the carboxylate group on the substrate Lys as substrate and H-Val-NH2 as added is blocked or not. This residue presumably nucleophile (33). Under these conditions the corresponds to the essential histidyl residue in acyl-enzyme intermediate is partitioned be- the serine endopeptidases, and as mentioned in tween hydrolysis and aminolysis products, i.e. section 5.1 a histidyl residue is indeed essential Z-Lys-OH and Z-Lys-VaI-NH2, respectively (see to the action of carboxypeptidase Y. Accord~ section 6.3). In case the deacylation reaction ingly, the high activity of the serine carboxypep- were rate-limiting, k~a, for the disappearance of tidases in the acidic pH ranges relative to the substrate should increase by the addition of corresponding endopeptidases can be explained H-VaI-NH2. However, no variation of k~a, was by unusually acidic essential histidyl residues. A observed, suggesting that the rate-limiting step number of serine carboxypeptidases have been lies ahead of the deacylation step, i.e. acylation reported to exhibit optima towards peptides is rate-limiting (k2,~k3). The acylation is as- significantly below that of malt carboxypeptid- sumed to be rate-limiting for all N-blocked ase II (e.g. 115, 141, 142, 143). However, the amino acid esters with the exception of sub- substrates used in these studies contain side- strates characterized by an unusually low k3, e.g. chains which ionize in this pH range, e.g. Z-Glu -$ trimethylacetyl nitrophenylester (51). Since the Tyr-OH, and accordingly the observed pH pro- rate of acylation for ester substrates is much files do not necessarily reflect ionizations on the faster than for amide substrates the acylation enzyme. step is also assumed to be rate-limiting for the

96 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM: Serine carboxypeptidases

I P1 P1 Leaving group I ! I ?,o ?,oI II II Y- NH-CH-C- NH-CH-C-OH Amino acid (a)

I II II Y - NH - CH - C - O - CH - O - OH Hydroxyacid (b)

Y - NH - CH - C - NH 2 Ammonia (c)

Y - NH - CH - C - NH - CH - C - NH 2 Amino acid amide (d)

Y- NH-CH-C-O- X Alcohol (e)

Scheme 5. Substrates of the serine carboxypeptidases. Y = peptidyl or N-blocking group; R, and R', = amino acid side-chain; X = Me, Et or Bzl.

hydrolysis of N-blocked dipeptides, and conse- hydrolysis reactions may formally be considered quently k,at - k2 and Km- K~ (see Scheme 4). as transacylation reactions with a peptide as acyl-donor, water as acyl-acceptor, and an amino acid as leaving group. However, like the serine endopeptidases, the serine carboxypep- 6. BINDING SITES IN SERINE tidases also catalyze reactions with other acyl- CARBOXYPEPTIDASES donors and acyl~ e.g. various alcohols 6.1. The influence of the substrate structure and amines. Thus, two types of specificity must The influence of systematic alterations in the be considered: the specificity in the acylation structure of the substrate on the kinetic parame- reaction towards the acyl-donor which is the ters for its hydrolysis is a commonly used meth- substrate, and the specificity in the deacylation od to obtain information about the nature of the reaction towards the acyl-acceptor, i.e. the nu- binding sites (reviewed by SVENDSEN (178)). cleophile. The present section describes only The degree of complementation between en- hydrolysis reactions and hence only the specif- zyme and substrate is perhaps best reflected by icity towards the acyl-donor. kc,,/Km since proteolytic enzymes as discussed by Carboxypeptidase Y and malt carboxypeptid- FRUTON (56) and FERSHT (54) appear to use ases I and II have in the present laboratory been part of the binding energy to increase k,a, and shown to catalyze the hydrolysis of substrates of consequently the value for K, (see Scheme 4) is the types listed in Scheme 5 (23, 28, 37, 98), and not necessarily a true measure for the affinity of it is probable that other serine carboxypeptidases the enzyme for different substrates. which are less extensively characterized have Carboxypeptidases are characterized by their similar specificities. It is apparent that serine inability to hydrolyse any other peptide bond carboxypeptidases do not exhibit an absolute than the C-terminal in a polypeptide chain. Such requirement for a carboxylate ion on the leaving

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 97 K. BREDDAM; Serine carboxypeptidases

Table IV. Influence of the leaving group on the kinetic parameters for carboxypeptidase Y catalyzed hydrolysis reactions.

Substrate pH kca, K~ k~,/K~ ref. (Scissile bond) (min-') (mM) (min-' mM-~) l Bz-GlyZPhe-OH 6.5 230 7.7 30 70,73 Bz-GIy-OGly-OH 6.5 1800 56 32 a~ Bz-GIy-OPhe-OH 6.5 1100 0.05 22000 98 Bz-GIy-OMe 7.5 150 48 3.1 a~ Bz-Gly-OEt 7.5 150 21 7.1 a) Bz-Gly-Oipr 7.5 260 20 13 ~1 Bz-Gly-OBzl 7.5 1 I00 1.7 650 ~1

Bz-AIa-OGIy-OH 6.5 3500 14 250 a~ Bz-AIa-OMe 7.5 3100 6.3 490 27 Bz-Ala-OBzl 7.5 7400 0.07 106000 27

Bz-Phe-Gly-OH 6.5 690 1.1 630 a~ Bz-Phe-OGly-OH 6.5 2100 2.4 880 ~ Bz-Phe-OMe 7.5 9100 0.18 51000 27

Z-Phe-GIy-OH 6.5 8400 4.0 2100 70, 73 Z-Phe-NH2 8.0 320 10 32 8 Z-Phe-NH-CH3 7.5 not hydrolysed 70, 73

FA-Phe-GIy-OH 6.5 5800 5.4 1100 34 FA-Phe-AIa-OH 6.5 9700 0.14 61000 34 FA-Phe-Val-OH 6.5 6500 0.047 140000 34 FA-Phe-Leu-OH 6.5 4900 0.021 230000 34 FA-Phe-NH2 7.5 - - 89 34 FA-Phe-Gly-NH2 7.5 - - 160 34 FA-Phe-VaI-NH2 7.5 - - 6200 34 FA-Phe-OMe 7.5 11000 0.39 28000 34 a) K.BREDDAM. unpublished results

group since they also, like the serine endopeptid- carboxypeptidases in further detail it is neces- ases, release alcohols and amines without a sary to analyze how the specificity constant negative charge. This is in contrast to the metallo kcat/Km is influenced by variations in each of the carboxypeptidases which are specific for pep- structural features which characterize the sub- tides and depsipeptides. Thus, although serine strate (see Scheme 5): a) the nature of the scissile carboxypeptidases do not cleave internal pep- bond, b) the importance of the negatively tide bonds they combine elements from the charged carboxylate ion of the leaving group, c) specificity of the serine endopeptidases with the the nature of the N-blockinggroup Y, and d) the specificity requirements of the metallo carboxy- nature of the amino acid side-chains RI and R~. peptidases. Nevertheless, carboxypeptidase Y The latter aspect is normally referred to as contains only a single active site since modifica- side-chain specificity, and for the sake of conve- tion of either of the essential seryl or histidyl nience the nature of X in alkyl esters is included residues abolishes the activities towards both in this concept. peptides and alkyl esters (see section 5.1). Table IV lists the influence of the leaving In order to analyze the specificity of serine group on the kinetic parameters for carboxypep-

98 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM:Serine carboxypeptidases tidase Y catalyzed hydrolysis ofamide and ester " $1 -'-~'- S~ )I bonds. Comparing substrates which only differ SCISSILE BOND in the nature of the scissile bond, one being an amide bond the other an ester bond reveals that the ester bond is hydrolysed with much higher I 9,' i o _,,f(o) rates than the amide bond. Thus, kcJKm for the P- NH-CH- C-NH-CH- C-O w% hydrolysis of Bz-GIy-J'OPhe-OH, an ester substrate with a carboxylate group on the leaving JI111111111111~]1]]]1]]]]]1~111111///111111]]]//]]1~ group, is 2-3 orders of magnitude higher than for / o IO Bz-Gly the corresponding peptide P- NH-CH- N., *[tb) substrate. For substrates without a carboxylate group on the leaving group similar differences .I /11/I/I/// are observed: k~=/K~ for the hydrolysis of FA- Phe is approximately 200 times higher I ,, than for the hydrolysis of FA-Phe and P-NH- ell- C- N H 2 +~(c) FA-Phe-~GIy-NH2. Similar observations have /II/I/I/1/ also been made with the malt carboxypeptidases and several serine endopeptidases. This influence of the scissile bond can be attributed to the higher reactivity of an ester bond as compared with an amide bond. The inherent strength of the amide bond where ammonia functions as leaving group is Figure 2. Schematic presentation of the binding re- similar to the strength of one where the leaving gions in the active site of carboxypeptidase Y. The binding notation is that of SCHECHTERand BERGER group is an amino acid. Nevertheless, as shown (163) (see abbreviations), and the dead-end structure of in this laboratory, kca,/Km for the carboxypeptid- the active site is assumed to account for the exo-pepti- ase Y catalyzed hydrolysis of FA-Phe-J'GIy-OHis dase specificity of carboxypeptidase Y. The figure 7-12 times higher than for FA-Phe-~NH2 and demonstrates conceivable binding modes of peptides, FA-Phe and for FA-Phe it is peptide amides, and peptide esters, accounting for the 23 times higher than for FA-Phe-J'Val-NHz (Ta- peptidase (a), peptidyl amino acid amide hydrolase (b), ble IV). This implicates a specific influence of amidase (c), and esterase (d) activities of the enzyme. the carboxylate group and one might expect that ester substrates would be similarly influenced by a carboxylate group such that kcJKm for the hydrolysis of depsipeptides would be much peptidase A Km for hydrolysis of peptides is higher than for corresponding alkyl esters. How- constant in the pH range 5 - 9 (3) consistent with ever, such an effect of a carboxylate group was tbearginylresidue(pKa- 12) being the binding not observed in all cases: Bz-Gly-J'OGly-OHand site for the C-terminal carboxylate group of the Bz-Gly are hydrolysed by carboxy- substrate ( 153, 157). Contrary to this, studies in peptidase Y with higher k~at/Krn than the corre- this laboratory indicate that Km for the carboxy- sponding alkyl ester, i.e. Bz-Gly and Bz- peptidase Y (34) and malt carboxypeptidase II GIy-OBzl, respectively, but Bz-Phe-OGly-OH (38) catalyzed hydrolysis of N-blocked dipep- and Bz-Ala are hydrolysed with lower tides increases sharply above pH 5.5 and pro- l~a,/K~ than the corresponding alkyl esters, i.e. vided that Km is a measure for K~ (see section 5.2) Bz-Phe and Bz-Ala-~OMe, respectively this pH dependence presumably eliminates an (Table IV). arginyl residue as binding site for C-terminal The observed effects of a C-terminal carbo- carboxylate groups. It is more likely that a xylate group in peptide substrates suggest the positively charged histidyl side-chain functions existence of a positively charged binding site on as anion binding site at acidic pH values. How- the enzyme for this group. In metallo carboxy- ever, the state of ionization of the proposed

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 99 K. BREDDAM"Serine carboxypeptidases

anion binding site has apparently no influence laboratory with malt carboxypeptidases I and II on the binding of ester substrates to these en- has demonstrated that the high I~,,IK~ for the zymes since K~ for the hydrolysis of esters is hydrolysis of Bz-Phe is mainly due to a essentially constant in the pH range 6-9. A high l~a, value while for Bz-Arg-~OMe it is mainly possible explanation could be that this binding due to a low Km value (30, 38). Detailed kinetic site is not in the proximity of the alcohol leaving studies with malt carboxypeptidase II indicate group of ester substrates but only "moves into that addition of NaCl has an adverse effect on the position" as a consequence of binding of a binding of FA-Arg-J'OMe while the binding of substrate with a charged C-terminal as is sug- FA-Phe appears tighter at higher ionic gested in Figure 2 which also illustrates how the strength suggesting ionic forces to be important various types of substrates might bind pro- for the interaction between binding site, and the ductively to the enzyme. positively charged side-chain of Arg, while hy- Kinetic studies with N-blocked dipeptides drophobic forces are responsible for the inter- and N-blocked amino acid esters have for some action with the uncharged side-chain of Phe serine carboxypeptidases provided information (38). These observations are consistent with a about the side-chain specificities, i.e. the influ- negatively charged residue on the enzyme being ence of R~, R~ and X on kcat/Km (Table V). important for the binding of positively charged Carboxypeptidase Y hydrolyses both ester and side-chains without having any particular influ- peptide substrates with aromatic and other hy- ence on binding of uncharged side-chains (see drophobic amino acid residues in the P1 position Figure 3). The results with malt carboxypeptid- much faster than those with hydrophilic amino ase I are qualitatively identical (30). The subtili- acid residues in this position. Thus, k,~/Km for sins have a similar wide specificity with respect the hydrolysis of Bz-Phe-~OMe is 4,000 times to the P~ position, and it has been suggested that higher than for Bz-Arg-~OMe, and similarly with this originates from different modes of pro- peptide substrates k,,/Km for the hydrolysis of ductive binding for substrates with an aromatic Z-Phe-~Ala-OH is 20,000 times larger than for and a basic residue in the P~ position (60). Bz-Lys (27). These results indicate that Other serine carboxypeptidases have not been the binding site for the side-chain at the P, studied in similar detail, but their substrate position is hydrophobic like that of chymo- preferences suggest that they exhibit specificities trypsin (see section 4). In contrast, the serine with respect to.the P, position corresponding to carboxypeptidases from watermelon, malt and that of either carboxypeptidase Y or to those of porcine kidney rapidly hydrolyse ester sub- the carboxypeptidases from watermelon, malt, strates both with hydrophobic and positively and human kidney. Human prolyl carboxypep- charged residues, i.e. Arg, Lys, or His, in the P~ tidase is an exception since it, due to its special position (Table V) (30, 38, 103, 129). Available function (see section 2), only cleaves substrates data for peptide hydrolysis by these enzymes where the P, position is occupied by a prolyl suggest a preference for hydrophobic amino acid residue. residues in the P~ position, but substrates with With the exception of malt carboxypeptidase positively charged residues have not been in- I1 all the enzymes listed in Table V exhibit cluded in systematic studies (103, 129). How- preferences for substrates with hydrophobic R'~ ever, it is conceivable that they all hydrolyse such (see Scheme 5) suggesting that they contain substrates with high rates as it has been demon- hydrophobic binding sites for this side-chain. strated for malt carboxypeptidases I and II (K. However, it should be noted that aliphatic hy- BREDDAM, unpublished results). Thus, the drophobic amino acids, i.e. Val, lle, Leu, Met, binding sites for the side-chain at the P, position and Ala, are released faster than aromatic resi- in the enzymes from malt, watermelon and dues, i.e. Phe and Tyr. The preference of malt porcine kidney are presumably more complex carboxypeptidase II for basic R~, i.e. Arg and than that ofcarboxypeptidase Y since they seem Lys, renders this enzyme unique among serine to accomodate an aromatic and a positively carboxypeptidases. Since it also releases hydro- charged side-chain equally well. Work in this phobic amino acid residues its binding site for

100 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM: Serine carboxypeptidases

A~ A~ A ~< O A < A A A A A e~ O < A A~ A O ~< .--.< e~ A A A ~2 A ^~, A ~ A A A A < A < A A ~ A A A d ~ < A 2^ A

O "~ Z

A eL r A A A A A A < A A~ tD A~ ^.~ L; O < A A A A A~ o L~ e~ A A 8~ ~A A < A ~} 3 A A A A"~ A .d A A . < -.1 A eL A~ A.~ A AN A_~ A A A ~> < A ~A < "~ Z

q o0

~ N

E 8~ < O

8o o o r~ ~ o8 ~'~.

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 101 K. BREDDAM: Serine carboxypeptidases

the side-chain at the P~ position presumably They concluded that the combination of an contains separate regions to secure the inter- unspecific substrate and an ammonium ion action with these groups of different nature. This simulates the behaviour of the specific substrate is supported by the fact that addition of NaCl has with a positively charged residue in the P~ an adverse effect on the binding ofFA-Ala-~Lys- position. Later work has demonstrated similar OH, whereas the binding of FA-Phe~Phe-OH is effects of numerous other amine and guanidine enhanced (38). This binding site therefore ap- compounds (162, 182, 189). pears similar to that for the R, group in both Among the serine carboxypeptidases three carboxypeptidases I and II. The influence of the cases have been reported where addition of a nature of X, i.e. the alcohol leaving group of ester substance produce activity changes which can be substrates, on 1~,, has only been investigated for related to its binding to particular substrate carboxypeptidase Y and malt carboxypeptid- binding sites. 3-phenyl-l-propanol increases La, ases I and II, and for these enzymes an increase and decreases Km for the carboxypeptidase Y in kcat/Kmis observed with increasing size (hydro- catalyzed hydrolysis of Z-Gly (6). Since phobicity) of the leaving group, suggesting that the hydrolysis of Z-Phe~Leu-OH, which has a the alcohol leaving group binds to hydrophobic bulky amino acid residue in the P~ position, is regions of the S~ binding site. inhibited by phenylpropanol this substance pre- It is known that carboxypeptidase Y hydro- sumably binds to the binding site for the side- lyses the substrates listed in Scheme 5 only when chain at the P~ position of the substrate. The the amino acid residues in the P, and P; positions hydrophobic nature of 3-phenyl-l-propanol are in the L-form (7, 70) and when the amino combined with the fact that the negatively group of the amino acid in the P~ position is charged 3-phenylpropionic acid does not bind at blocked, i.e. no hydrolysis is observed when Y = this binding site is consistent with it being H. k~a,/Km for carboxypeptidase Y catalyzed hydrophobic. hydrolysis of peptide, amide, and ester sub- The ability of malt carboxypeptidase II to strates is strongly affected by the nature of the hydrolyse substrates containing either hydro- N-blocking group occupying the P2 position and phobic or positively charged amino acid residues decrease in the following order: Z, Bz, FA, and on both the P, and P; positions combined with Ac (8, 27, 34, 70). It is unknown how the length the effects of added NaCI already suggested that of the peptide chain influences kcat/Km for the within the S, and S; binding sites separate areas hydrolysis of the C-terminal ester or amide exist to secure the interaction with these side- bond. chains of different nature (see section 6.1). A postulated binding region of the active site of malt carboxypepfidase II, incorporating this 6.2. Studies with modifiers hypothesis, is shown in Figure 3. It is assumed The effects on catalysis of substances which that the S~ and S~ portion of the S~ and S~ bind specifically to the binding sites, i.e. too- binding sites, respectively, contain negatively differs, have previously been utilized to study the charged groups which interact with the posi- nature of binding sites in proteolytic enzymes as tively charged side-chains of e.g. FA-Arg-~OMe illustrated by the work of INAGnMI and and FA-Ala The Sib and S~b portions, MURACHI on trypsin (93). This enzyme hydro- on the other hand, are assumed to be hydropho- lyses substrates with positively charged side- bic regions which provide the interaction with chains, i.e. Lys and Arg, in the P. position with hydrophobic side-chains of the substrate. It high rates but hydrolyses only very slowly sub- appears that phenylguanidine binds to the $1, strates with other amino acid residues in this and S]a binding sites of malt carboxypeptidase II position. However, in the presence of positively with an attenuation of the binding of FA-Arg-$ charged substances like methyl ammonium, OMe and FA-Ala as a result (38). In ethyl ammonium, or l-propylammonium ions contrast, the binding of uncharged substrates k~, for the hydrolysis of Ac-Gly-OEt is drastic- containing bulky groups in either the P~ or P] ally increased while K~ remains unchanged. positions, e.g. FA-PhekOMe, is enhanced in the

t02 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM:Serine carboxypeptidases S|a Slb Sio. Stb to hydrolyse substrates with basic residues in the P; position suggests the existence of only a single region in S; binding site. However, it appears that phenylguanidine does not bind to the S,a binding site of malt carboxypeptidase I since it enhances the rate of the hydrolysis of Bz-Arg OMe. Thus, in spite of apparent similarities in the S, binding sites of the two malt carboxypeptidases, as judged by their specificities, certain structural differences must exist to account for their different behaviour towards phenylguanidine. The apparent absence of an S;a binding site in carboxypeptidase I would predict that phenylguanidine should not bind to its S; binding site. However, in the presence of this substance the activities towards FA-Phe and FA-Phe~ Gly-NH2 are decreased to less than 3% of the I P1 I PI' I control whereas the activities towards FA-Phe Figure 3. Schematic representation of the binding OMe and FA-Phe are increased to 380% regions in malt carboxypeptidase II. The binding and 220% of the control, respectively. These notation is that of SCHECHTER and BERGER (see results indicate that when the leaving group of abbreviations), and the dead-end structure is assumed the substrate is large, i.e. an amino acid or an to account for the exo-peptidase activity ofthe enzyme. amino acid amide, it competes with phenyl- (A) shows a proposed binding mode of a peptide with guanidine for the same site. The activation lysyl residues in the P~ and P; positions. It is assumed observed with FA-Phe~OMe and FA-Phe-'tNH_, that the positively charged side-chains of Lys interact suggests that the binding of phenylguanidine with negatively charged groups located in the S,a and S;a affects the catalytic properties of the enzyme portions of the St and S; binding sites, respectively. (B) without adversely affecting its ability to bind shows a proposed binding mode of an analogous peptide with phenylalanyl residues in these positions. substrates with small leaving groups, i.e. the Here it is assumed that the hydrophobic side-chains enzyme may conceivably bind both groups si- interact with the hydrophobic S,b and S;b regions of the multaneously. A kinetic study of the effects of S, and S', binding sites, respectively. phenylguanidine on the hydrolysis of ester substrates with leaving groups of different size confirmed this interpretation (30). While k,, for the hydrolysis of all the substrates, i.e. Bz-Arg-~ presence of phenylguanidine. This suggests that OMe, -OEt, -OPr and -OBu, is increased by the such groups when bound to the S~b or S~b sites presence of this substance, Km is affected in a interact favourably with the phenyl group of the manner which depends on the size of the leaving phenylguanidine bound at the S,a or S;a sites. group: it decreases for Bz-Arg-~OMe and increas- When non-bulky groups are bound to both the es for Bz-Arg-~OEt, -OPr and -OBu. Thus, it S,b and S;b binding sites, viz. FA-Ala-~OEt, the appears that phenylguanidine binds to the bind- interaction with the phenylguanidine appears to ing site for the leaving group of ester substrates, increase k,, rather than to increase binding of the i.e. the S', binding site, and that this position in substrate. These results with phenylguanidine the enzyme functions as the binding site for the support the model of the binding region of malt leaving groups of all types of substrates. The carboxypeptidase II shown in Figure 3. effects of chemical modifications ofa methionyl The specificity of malt carboxypeptidase I (see residue located at the S; binding site of carboxy- Table V) suggests that its S, binding site com- peptidase Y on its specificity (see section 6.4) prises two distinct regions as indicated for malt indicates that this conclusion is valid for carbo- carboxypeptidase II in Figure 3, but its inability xypeptidase Y as well.

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 103 K. BREDDAM"Serine carboxypeptidases ,~k3 E§ [P3]/[P2], is a linear function of the ratio [N]/[W] between the concentrations of added nucleo- Ks k2 [w] phile and water. E+S ~ ES > ES m Carboxypeptidase Y and the malt carboxy- + k~[N] peptidases have similarly in this laboratory been demonstrated to catalyze transacylation reac- PI E+P 3 tions to other nucleophiles than water, e.g. to alcohols, amino acids and amino acid deriva- dP2/dt = k3[W][ES' ] tives(24, 33, 38, 195, 196, 197). For carboxypeptidase Y it has been shown, using N-blocked amino acid esters as substrates, that the fraction dP3/dt : k4[N][ES' ] ofaminolysis, P3/(P2+P3), is highly dependent on the nature of the amine nucleophile added. With P3 k4[N] H-Asp-OH, H-Asp-a-OMe, H-Asp-ct-NH2, H- Glu-OH, H-Glu-tt-OMe, H-GIu-et-NH2, H-Pro- P2 k3[W] OH, H-Pro-OMe, and H-Pro-NH2 no amino- Scheme 6. Competition between water (W) and other lysis is observed. However, with the other L- nucleophile (N) in the serine protease catalyzed hydro- amino acids, amino acid methyl esters, and lysis of ester substrates as proposed by BENDERet al. amino acid amides aminolysis is observed with (12). The enzyme (E) and substrate (S) initially form a the fraction of aminolysis varying with the complex (ES) with the dissociation constant Ks. k2 is the nature of the side-chain as seen from Table VI. rate constant for the essentially irreversible conversion D-amino acids, D-amino acid methyl esters, and of ES complex to the acyl-enzyme intermedidate (ES'). D-amino acid amides do not react with the k3 and k4 are the rate-constants for the deacylation acyl-enzyme, suggesting that the reaction with reactions with W and N as nucleophiles, respectively. the nucleophile is stereospecific. No aminolysis PL is the alcohol leaving group of the ester substrate. P2 takes place with secondary amines, e.g. proline and P3 are the hydrolysis and aminolysis products, respectively. This model assumes that neither water and sarcosine, and more bulky substituents on nor added nucleophile bind to the enzyme prior to the the carbonyl group than -OMe and -NH2, e.g. reaction with the acyl-enzyme intermediate. -OEt, -O'Bu, and -NH-CH3 result in decreased fractions of aminolysis with the exception of H-Gly-OEt and H-GIy-O~Bu. The variations in the efficiency of the nucleo- 6.3. Binding sites for nucleophiles philes listed in Table VI cannot be attributed to In the serine protease catalyzed hydrolysis of different nucleophilicities. This combined with amide and ester bonds water functions as nu- the stereospecificity of the enzyme towards the cleophile. When such reactions are allowed to nucleophile would suggest that binding of the proceed in aqueous solution with another nu- added nucleophile to the acyl-enzyme inter- cleophile added, e.g. an amine compound, a mediate, presumably at the S~ binding site, partitioning of the acyl-enzyme may take place preceeds its participation in the deacylation to yield a hydrolysis product and an aminolysis reaction. In fact, the results listed in Table VI product. BENDER et al. (13) and FASTREZ and suggest that the nucleophiles which react with FERSHT (53) found that when methanol, etha- the acyl-enzyme intermediate exhibit structural nol, glycine amide or alanine amide were added resemblance with the leaving groups (P;) of the as nucleophiles to chymotrypsin it reacted with substrates described in Scheme 5. However, the ester substrates according to the simple reaction fact that proline is released from the C-terminus model shown in Scheme 6, in which it is as- of peptides by carboxypeptidase Y and never- sumed that neither water nor the added nucleo- theless cannot function as nucleophile in the phile bind to the enzyme prior to the reaction deacylation reaction suggest that certain diffe- with the acyl-enzyme intermediate. Hence, the rences between the structures of substances ratio of the two reaction products being formed, which can function as leaving groups and those

104 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM:Serine carboxypeptidases

Table VI. Carboxypeptidase Y catalyzed aminnlysis aminolysis reactions were often performed at reactions. arbitrary concentrations of nucleophiles, and Fraction of from such results it is not possible to decide Nucleophile aminolysis whether a high fraction of aminolysis with a given nucleophile is due to a tight binding of the nucleophile to the enzyme or alternatively, due 0. l0 - 0.65 to an effective attack of the nucleophile on the acyl-enzyme intermediate. Malt carboxypeptid- Substitutions on carbonyl group: ase I similarly catalyzes aminolysis reactions of ester and peptide substrates when various amino H~N-~-~-OMe 0.25-0.90 acid amides and methyl esters are used as nucleophiles, and these reactions have now been H2N-~-~-O~Bu ~ 0 -0.10 investigated in further detail (33) than the corresponding reactions with carboxypeptidase Y. H2N-~-~-NH: 0.70-0.90 The influence of the concentration of nucteo- phile on the fraction ofaminolysis demonstrated H2N-~-~-NH-CH3 0.10 that the enzyme was easily saturated with nucleophile indicating the formation of a complex 0 between nucleophile and acyl-enzyme inter- Substitutions on amino group: mediate prior to deacylatio a. This observation is consistent with the fact that phenylguanidine, HN-C-C-OH which has been shown to bind to the S; binding site of malt carboxypeptidase I (see section 6.2), when added to aminolysis reactions causes a HN~-Ci-~-OH (proline) drastic increase in KN(app),the apparent dissociation constant of the complex between acyl-en- All reactions are performed with Bz-Ala-~OMeas sub- zyme and the tested nucleophiles. This indicates strate at pH 9.5 using various concentrations ofnucleo- a competition between phenylguanidine and the phile. R = amino acid side-chain with the exception of nucleophiles for the S~ binding site of the en- the side chains of Asp and Glu. Fraction ofaminolysis is defined as P3/(P2+P3) with P2 = hydrolysis product zyme. Phenylguanidine has no effect on the and P3 = aminolysis product (see Scheme 5). ~ H-Gly- aminolysis reactions with H-Gly-OMe but O~Bu is an exception since the fraction of aminolysis strongly affects KN(app) for H-AIa-OMe and H- with this nucleophile is as high as with the methyl ester. VaI-OMe suggesting that it is the side-chains of Results from ref. 196 & 197. amino acid methyl esters which are bound to the S~ binding site in such a manner that they occupy or overlap with the binding site for phenylguanidine. No such differences were observed which can function as nucleophiles do exist. with amino acid amides: phenylguanidine af- Althougt~ the release of an amino acid ester from fected KN(,,,) for all amino acid amides tested a peptide ester would be difficult to demonstrate including H-GIy-NHz. This indicates that ester due to the much faster release of the alcohol and amide nucleophiles exhibit slightly different group it should be noted that this reaction has binding modes within the S~ binding site of the not been demonstrated and yet, amino acid enzyme. This could explain why amino acid methyl esters can function as nucleophiles. amides are more effective as nucleophiles than The studies of carboxypeptidase Y catalyzed amino acid methyl esters, viz. the fraction of aminolysis reactions were originally aimed at aminolysis at saturation with nucleophile is the synthetic aspects of such reactions rather much higher with amino acid amides (0.90) than than investigating in detail the causes for the with amino acid methyl esters (0.50). It may also different fractions of aminolysis with nucleo- explain why for amino acid amides KN(~,p) is philes of different structures. Consequently, the lower and less dependent on the nature of the

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 105 K. BREDDAM:Serine carboxypeptidases amino acid side-chain than for amino acid modifications of essential amino acid residues methyl esters. However, the binding of both usually abolish the catalytic activity, modifica- types ofnucleophiles appears to be stereospecific tions of side-chains at binding sites may have since with the D-enantiomers H-val-NH2 and different effects on activity, depending on the H-ala-OMe no aminolysis products are ob- nature of the reagent and the importance of the tained. particular side-chain as previously demon- FINK and BENDER (55) have previously ob- strated with some proteolytic enzymes (44, 80, served similar binding effects when larger n- 178). Recently, it has become possible by site-di- alkyl alcohols were added to papain acting on rected mutagenesis to specifically exchange p-nitrophenyl ester substrates. They found that amino acid residues in proteins (206). Modifica- their results could only be explained by adopting tions of this type are in many ways less drastic a reaction scheme in which the added nucleo- than the chemical modifications provided that phile binds to the enzyme at two different the mutated enzyme is able to attain its proper positions, one of which is identical to the binding tertiary structure. Both chemically modified and site for the leaving group, i.e. the S~ binding site. mutated enzymes can provide insight into the By studying the influence of H-VaI-NH2 on the nature of the binding sites beyond what is steady-state parameters, k~,, and Km, for the obtained by kinetic studies and in addition, these action of malt carboxypeptidase on N~t-CBZ- may have an altered specificity which is of Lys-ONp it was demonstrated that this enzyme potential interest in processes where proteolytic also has two binding modes for the added nu- enzymes are used (see section 7). cleophile, of which only one causes aminolysis of the acyl-enzyme intermediate (33). More recently RIECHMANN and KASCHE have found 6.4.1. Modifications at the S, binding site that nucleophiles similarly bind to chymo- It has long been known that carboxypeptidase trypsin prior to their participation in the deacyl- Y from baker's yeast contains a cysteinyl residue ation reaction (156), suggesting that such bind- (71) and this has recently, in this laboratory, ing is general for proteolytic enzymes. The been identified in the amino acid sequence as results with malt carboxypeptidase I (33) dem- Cys-341 (36). This residue is inaccessible to onstrate that studies of the competition between iodoacetate and EUman's reagent unless the water and added nucleophiles in the deacylation enzyme is denatured. However, without denatu- reaction may provide information about the ration of the enzyme it reacts rather slowly with binding sites of proteolytic enzymes which can- p-HMB (k = 0.45 mM -~ sec-') producing the not be obtained from the study of hydrolysis spectral change expected for a cysteine and it reactions. reacts quickly with Hgt+ (k = 1000 mM t sec t) (6). Only stoichiometric amounts ofp-HMB or Hg "+ are bound, and Hg" apparently binds 6.4. Modifications of binding sites tighter to the sulfhydryl group than the mercuri- Chemical modifications of amino acid side- benzoate group since addition of 1 equivalent chains in enzymes have been used in numerous Hg§ to carboxypeptidase Y previously modified cases to correlate lo,~s of a certain amino acid with p-HMB results in an instantaneous dis- residue with changes in activity in order to placement of the mercuribenzoate group from establish which side-chains are functionally es- the cysteinyl residue. The different reactivities of sential. In such studies it is important to mercurials and iodoacetate may be due to diffe- distinguish between two types of side-chains: a) rent abilities to penetrate the enzyme molecule those which are essential to catalysis (see section before reaction with the sulfhydryl group or 5.1) and b) those which alone or in combination alternatively, due to different abilities to pro- with others function in the binding of substrate, duce a conformational change rendering the e.g. the side-chains in chymotrypsin which form buried cysteinyl residue accessible. the hydrophobic crevice for the side-chain in the Modification of the cysteinyl residue with P, position of the substrate. While chemical organomercurials causes significant inhibition

106 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM:Serine carhoxypeptidases

Table VII. Relative specificity of carboxypeptidase Y and carboxypeptidase Y modified with I-Hg ~ and Ph-Hg*. Substrate CPD-Y 1-Hg-CPD-Y Ph-Hg-CPD-Y Bz-Gly-OMe 1 1 1 Bz-Ala-OMe 160 89 43 Bz-Val-OMe 58 111 4 Bz-Ile-OMe 84 360 2 Bz-Leu-OMe 1800 1700 68 Bz-Met-OMe 900 6100 30 Bz-Phe-OMe 16000 17000 790 Bz-Lys-OMe 0.18 46 0 Bz-Arg-OMe 3.9 30 0 Bz-His-OMe 4.2 21 0 Bz-Thr-OMe 5.8 13 0.30 The numbers which represent relative k,,/Km values can only be compared within the same column. The results are from ref. 27.

of the enzyme, but this residue is nevertheless BAI and HAYASHI (6) found that the inhibi- not essential for catalysis since substantial activ- tion ofcarboxypeptidase Y by organomercurials ity is retained towards some substrates (6, 27), is most pronounced when the enzyme is assayed and the pH dependence ofk~, for ester hydrolysis towards substrates with bulky side-chains in the remains unchanged (27). This is consistent with Pj position, indicating that the sulfhydryl group recent experiments where Cys-341 has been is located at the binding site for this side-chain. exchanged by site-directed mutagenesis with a This information warranted an extensive study seryl residue or a glycyl residue. These deriva- by the present author of the influence of modifi- tives of carboxypeptidase Y are active, but the cation with mercurials on the specificity of activity towards peptide substrates is only carboxypeptidase Y with respect to the P~ posi- around 10% (J. WINTHER, M.C. KIELLAND- tion (27). The kinetic constants for a series of BRANDT and K. BREDDAM, unpublished re- ester substrates with the general formula Bz-X sults). OMe, where X = amino acid residue, have been Studies in this laboratory have shown that determined for unmodified carboxypeptidase Hg ++, in the absence ofhalides, i.e. Cl-, Br-, SCN-, Y, carboxypeptidase Y modified with I-Hg~ and CN-, causes complete inactivation ofcarbo- (Hg +* modified enzyme assayed in the presence xypeptidase Y regardless of the nature of the of I-), and carboxypeptidase Y reacted with substrate (27). This total lack of activity of the phenylmercuric chloride. Modification with modified enzyme is possibly due to the mercuric both mercurials causes a decrease in k~ol and an ion forming a -S-Hg-X-bridge between the sulf- increase in Km for the hydrolysis of all substrates hydryl group and some other amino acid side- tested with the exception of Bz-Lys-~OMe where chain X capable of forming a rather tight com- modification ofI-Hg~ caused a 40 times increase plex with Hg *§ e.g. X = His, Asp, or Glu (194). in k~at. A comparative assessment of the specif- This explanation is supported by the obser- icity of these three enzymes has been made by vation that addition of halides which form tight calculating relative kcat/Km values with Bz-Gly-+ complexes with Hg +* (194), partially reactivates OMe serving as reference for each enzyme (Ta- the enzyme without removing the metal from ble VII). As previously mentioned carboxypep- the enzyme (27). It is conceivable that this is due tidase Y exhibits a strong preference for sub- to the halide displacing X from the mercuric ion strates with hydrophobic residues in the Pt bound to the sulfhydryl group thus forming an position, i.e. Phe, Leu, and Met, and only slowly active enzyme species, e.g. -S-Hg-C1 when C1- is hydrolyses substrates with hydrophilic residues, added. i.e. Arg, Lys, Thr, and Gly. The two modified

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 107 K. BREDDAM:Sefine carboxypeptidases enzymes retain the highest kcat/Kmfor substrates known, but the essential histidyl residue is a with hydrophobic residues although their rela- possibility since the histidyl side-chain is known tive preference for the different substrates to form complexes with Hg+* (194). In this deviate substantially and differ from the prefer- context it should be noted that CHAMBERS et at. ences of unmodified enzyme. With carboxypep- (42) by X-ray crystallography have demon- tidase Y modified with Ph-Hg§ the relative strated that Ag+ binds to a position between the k~a,/Km is reduced drastically for all substrates aspartyl and histidyl side-chains of the catalytic except Bz,Ala This is equivalent to an triad in trypsin, and Hg++ might be expected to increased preference for Bz-GIy and Bz- have a similar affinity for this position. Ala and can be explained by the Ph-Hg+ The influence of rig +§ on malt carboxypeptid- group introducing a steric hindrance into the ase I which does contain a sulfhydryl group is binding site for the side-chain at the P~ position essentially identical to that on malt carboxypep- with the consequence that the hydrolysis of tidase II (28) and thus, it is probable that the substrates characterized by a bulky side-chain in inactivation is not due to reaction with the the Pj position, e.g. Bz-Phe-COMe, is much more sulfhydryl group. This is consistent with this affected by the Ph-Hg~ modification than the group being inaccessible to p-HMB as opposed hydrolysis of substrates with non-bulky side- to the sulfhydryl group in carboxypeptidase Y chains in this position, i.e. Bz-Gly-~OMe and (27). Dilution of malt carboxypeptidase I inac- Bz-Ala Introduction of the I-Hg* group tivated by Hg++ apparently causes a dissociation has not similar steric effects since the hydrolysis of the enzyme-Hg"§ complex. However, the of Bz-Gly-~OMe and Bz-Phe-~OMeare affected to activity of the enzyme was only partially recov- the same extent, i.e. the relative k~at/Km for ered and with increasing reaction period a de- Bz-Phe-J'OMeremains unchanged. However, the creasing proportion of the enzyme could be drastic increase in the relative kcat/Km for the reactivated. These results indicate that the initial hydrolysis of Bz-Lys-~OMe and the slight in- enzyme-Hg§247 complex undergoes an irreversible crease for substrates with Val, Ile, Met, Arg, His, time dependent inactivation presumably due to and Thr in the P, position indicate that the denaturation of the enzyme. The time-course of properties of the binding site of the modified this denaturation suggests a complex reaction enzyme is different from that of the unmodified scheme which might involve conformational enzyme. The exact nature of this change is changes as well as a secondary reaction of Hg§247 difficult to evaluate; possibly the effects of the with the sulfhydryl group. I-Hg+group on Bz-Lys-~OMehydrolysis are due p-HMB inhibits a number of the serine carbo- to attenuation of an unfavourable interaction xypeptidases from fungi (Table I) and the vari- between enzyme and substrate. ous types ofcathepsin A (Table Ill) and hence, it Malt carboxypeptidase II contains no sulfhy- is probable that these enzymes contain a sulfhydryl group but it is still inactivated by Hg~ (37). dryl group of reactivity as the one in carboxypep- However, while the carboxypeptidase Y-Hg+ tidase Y. The carboxypeptidases from higher complex exhibits a dissociation constant far plants (Table II) and human proly[ carboxypep- below 108 M in the pH range 4 - 10 (K. tidase, on the other hand, are not inhibited by BREDDAM, unpublished results) the formation p-HMB. An inhibitory effect of Hg§ is often of a malt carboxypeptidase II-Hg§ complex is seen with these enzymes but as shown with the dependent on deprotonation of a group on the malt carboxypeptidases this does not necessarily enzyme with an apparent pKa of 6.4 and the indicate the presence ofa sulfhydryl group. dissociation constant is rater high (1.3- 10.6 M at The serine endopeptidases protease B from pH 7.5). The comparatively poor binding of yeast (106) and thermitase from Thermoactino- Hg++ to carboxypeptidase II is also apparent myces vulgaris (10) also contain a single sulfhy- from the lack of inhibition in the presence of dryl group. Both enzymes are inhibited by p- iodide since this indicates that Hg ++ binds less HMB and Hg§ but not by other reagents known tightly to the enzyme than to iodide. The site of to modify thiols indicating that their sulfhydryl reaction in malt carboxypeptidase 1I is un- group is buried and of similar accessibility as the

108 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM:Serine carboxypeptidases one in carboxypeptidase Y. A peptide contain- KUHN et al. (113) have described that alkyla- ing the cysteinyl residue of thermitase has been tion ofa methionyl residue in carboxypeptiase Y shown to exhibit homology with a stretch of the with iodoacetamide causes a reduction in the amino acid sequence of subtilisin. The position peptidase activity of the enzyme without signi- of the cysteinyl residue in thermitase corre- ficantly altering the esterase activity. In this sponds in subtilisin to Val-68 which in the laboratory, this methionyl residue has been three-dimensional structure is located behind identified as Met-398 in the amino acid se- the histidyl residue of the catalytic triad. The fact quence (36) and in addition it has been alkylated that so many genetically unrelated but functio- with phenacylbromide, m-nitrophenacylbrom- nally similar enzymes contain a sulfhydryl group ide and p-nitrophenacylbromide (34) and oxid- suggests that it has a function although it is not ized with H202 (35). All these derivatives of strictly required in catalysis as indicated by the carboxypeptidase Y have been separated from studies with carboxypeptidase Y (6, 27). Possi- residual unmodified enzyme by affinity chro- bly, it could have a function in stabilizing the matography and characterized kinetically (34, protein structure since carboxypeptidase Y is 35). Modification of carboxypeptidase Y with destabilized by chemical modification of the phenacylbromide influences KdK~ for the hy- sulfhydryl group (27) and by replacement of the drolysis of ester and amide substrates in a cysteinyl residue with a glycyl or seryl residue by manner which is dependent on the size of the site-directed mutagenesis (J. WINTHER, M.C. leaving group of the substrate (Table VIII). With KIELLAND-BRANDT, K. BREDDAM, unpub- substrates containing a non-bulky leaving lished results). group, i.e. -OMe and -NH2, k~a,/Kmis increased while it is decreased for substrates with larger leaving groups, i.e. -OEt and -Gly-NH2, and in particular for those with very bulky leaving 6.4.2. Modification at the S; binding site groups, i.e. -OBzl and -Val-NH2. These results The S'~ binding site in serine carboxypeptid- suggest that Met-398 is located in the S~ binding ases presumably consists of binding sites for both site, and presumably in the binding pocket for the side-chain and the carboxylate group of the the side-chain of the C-terminal amino acid C-terminal amino acid residue of the substrate residue since the hydrolysis of FA-Phe-~Val-NH2 (see Figure 2). KUHN et al. (113) have, based on is much more affected by the modification than chemical modifications with phenylglyoxal, the hydrolysis of FA-Phe-~GIy-NH2. This sug- suggested that an arginyl side-chain functions as gests that bulky leaving groups of ester substrates the carboxylate binding site in carboxypeptidase bind to this position as well. Similar activities Y, but results obtained in the present laboratory were obtained with carboxypeptidase Y alky- on chemical modifications of carboxypeptidase lated with m-nitrophenacylbromide and p-ni- Y with phenylglyoxal and butanedione have not trophenacylbromide. The enzymes in which the provided evidence for this conclusion (21). Malt same methionyl residue has been alkylated with carboxypeptidase I has also been treated with iodoacetamide to form a non-bulky methionyl these reagents and no inactivation of this en- sulfonium derivative (34) or oxidized with H202 zyme was observed (K. BREDDAM, unpublished to form a methionyl sulfoxide derivative (35) results). In addition, kinetic studies with both hydrolyse all ester and amide substrates, includ- carboxypeptidase Y and malt carboxyptidase II ing those containing small leaving groups, at have suggested that the binding of peptides is reduced rates relative to unmodified carboxy- dependent on the deprotonation of an ionizable peptidase Y. Thus, it appears that the modified group with a pK~ around neutral (see section 6.1) enzyme hydrolyses substrates with small leaving which is far below the pKa of an arginyl side- groups at higher rates than unmodified CPD-Y chain, but instead suggests the involvement of a only when a bulky hydrophobic group, e.g. the histidyl residue or, alternatively, the N-terminal phenacyl group, is introduced into the S'~binding a-amino group in the binding of peptide carbo- site. xylate groups. Alkylation of Met-398 with phenacylbromide

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 109 K. BREDDAM: Serine carboxypeptidases

Table VIIL Hydrolysis of ester, amide and peptide substrates by carboxypeptidase u and pbenaeylbromidemodified carboxypeptidase Y.

Substrate Enzyme kcat Km KadK~. (min") (raM) (min". mM-~) CPD-Y 11000 0.39 28000 FA-Phe-~OMe PAB-CPD-Y 6400 0.078 82000 CPD-Y 4000 0.36 11100 FA-Leu-~OMe PAB-CPD-Y 2300 0.041 56000 CPD-Y 11000 0.059 190000 FA-Phe-~OEt PAB-CPD-Y 4500 0.1 ! 41000 CPD-Y 4600 1.0 4600 FA-AIa-~OEt PAB-CPD-Y 30000 14 2100 CPD-Y 9100 0.054 170000 FA-AIa-~OBzl PAB-CPD-Y 680 0.43 1600

CPD-Y 0.95 FA-VaI-~NH2 PAB-CPD-Y 1.7 CPD-Y 52 FA-Leu-~NH_, PAB-CPD-Y 160 CPD-Y 89 FA-Phe-~NH., PAB-CPD-Y 220 CPD-Y 160 FA-Phe-~GIy-NH2 PAB-CPD-Y 78 CPD-Y 6200 FA-Phe-+VaI-NH2 PAB-CPD-Y 71 CPD-Y 5800 5.4 1100 FA-Phe-~GIy-OH PAB-CPD-Y 180 1.6 110 CPD-Y 9700 0.14 61000 FA-Phe-~Val-OH PAB-CPD-Y 260 0.42 620 CPD-Y 4900 0.021 230000 FA-Phe-~Leu-OH PAB-CPD-Y 41 0.033 1200 The results are from ref. 34.

110 Carlsberg Res. Commun. Vol. 51, p, 83-128, 1986 K. BREDDAM: Serine carboxypeptidases reduces k~, for the hydrolysis of all peptides to bic leaving groups, Km is decreased. Correspon- approximately 2% of the values obtained with dingly, Leu-398-CPD-Y exhibits decreased 1~,/ unmodified CPD-Y while Km is only slightly I~ values towards substrates with small leaving increased (Table VIII). The small influence of groups, and increased values for the substrates the alkylation on Km suggests that the postulated with large leaving groups. The apparent tighter positively charged binding site which is expected binding to Leu-398-CPD-Y of substrates with to function when the enzyme forms tight com- bulky and hydrophobic P; groups is in accord- plexes with peptide substrates (see section 6.1) is ance with the leucyl side-chain being slightly unaffected by the alkylation of the methionyl smaller and more hydrophobic than the methio- residue. This is consistent with the pH depend- nyl side-chain. The increased preference for Phe ence of Km for peptide hydrolysis remaining in the P~ position is essentially independent of unchanged by the modification with phenacyl- the amino acid residue occupying the P, position bromide relative to unmodified carboxypeptid- (199) and thus, it appears that only the specif- ase Y (34). The drastic reduction in kca t is icity with respect to the P~ position is affected by unlikely to be due to the loss of Met-398 since the Met to Leu mutation which is consistent oxidation of this residue only reduces Ka, for with Met-398 being located in the S] binding site. peptide hydrolysis to 35 - 50% of the values Arg-398-CPD-Y exhibits kinetic parameters obtained with unmodified CPD-Y (35). Fur- similar to those obtained with the enzyme in thermore, the derivative alkylated with iodoa- which Met-398 has been modified with iodoacetamide hydrolyses peptides with kinetic cetamide and this is consistent with the parameters similar to those of the derivative structural resemblance between an arginyl resi- alkylated with phenacylbromide indicating that due and this particular methionyl sulfonium the bulkiness of the group introduced only has derivative (L. BECH, J. WINTHER, M.C. KIEL- little influence. It is more likely that the sulfo- LAND-BRANDT, K. BREDDAM, unpublished re- nium ion introduced into all the alkylated de- suits). rivatives is the cause of the reduction in k~a,, and Treatment of carboxypeptidase Y with H202 it may be speculated that this group prevents a in acetate buffer rather than in phosphate buffer conformational change which might be essential converted both Met-398 and Met-313 to the for the high k~ values characteristic for hydro- corresponding sulfoxide derivatives (35, 36). lysis ofpeptides. The fact that hydrolysis of ester The influence of the buffer on the oxidation and amide substrates is adversely affected only reaction is presumably due to acetic acid in the when the leaving group of the substrate is bulky presence of H202 being converted to peracetic would then suggest that the postulated confor- acid, a much more potent oxidizing agent than mational change is not essential for the hydro- H202 itself. The enzyme with both Met-313 and lysis of substrates with blocked C-terminus. Met-398 oxidized is, relative to the enzyme with To further study the role of Met-398 this only Met-398 oxidized, characterized by 2-4 fold amino acid residue has been substituted by a higher activity towards all substrates. It has not leucyl residue or an arginyl residue by means of yet been possible to establish whether this in- site-directed mutagenesis (199). Compared with crease in activity observed upon modification of carboxypeptidase Y, Leu-398-CPD-Y generally Met-313 is due to this residue being located in a exhibits unchanged or moderately reduced l~a, particular binding site. However, it should be values for the hydrolysis of peptide and ester noted that while oxidation of Met-398 has very substrates, whereas K~ is affected in a way which little influence on the thermal stability ofcarbo- is dependent on the group occupying the P] xypeptidase Y, the additional oxidation of Met- position. For the substrates FA-Phe~Gly-OH, 313 causes a drastic decrease in the stability (35). FA-Phe and FA-AIa-~OEt, which all are Treatment ofcarboxypeptidase Y with fluoro- characterized by relatively small groups at the P~ dinitrobenzene resulted in the specific modifica- position, K~ is increased, while for FA-Phe-~Leu - tion of a tyrosyl residue, forming a DNP-O-Tyr OH, FA-Phe and FA-AIa-~OBzl, all derivative (35). The specificity of this modified characterized by bulky and strongly hydropho- enzyme suggests that this particular tyrosyl resi-

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 111 K. BREDDAM"Serine carboxypeptidases

I l I | I I 8 1 I I /

._~6 ~n~exo

0 2.0 40 60 MINUTES 0 50 100 Figure 4. Digestion of oxidized ribonuclease with MINUTES carboxypeptidase Y. Reaction conditions: 0.5 mM oxidized ribonuclease, 0.1 M-Mes, pH 7.0, 0.27 pM- Figure 5. Digestion of oxidized ribonuclease with CPD-Y. For the amino acids released from the C-ter- carboxypeptidase Y. Reaction conditions: 0.5 mM minus the followingsymbols were used: Val, O; Ser,e; oxidized ribonuclease, 0.1 M-Mes, pH 5.0, 0.8 IXM-car- Ala,x; Asp,rq; Phe,A; His,A; Pro,! boxypeptidase Y. The symbols are listed in Figure 4.

due is located in the S; binding site. However, its peptides which are otherwise insoluble. How- position in the amino acid sequence has not yet ever, in 6 M-urea carboxypeptidase Y is inacti- been identified. vated with time, e.g. at pH 7.0:t,/2 = 2 hours, and for this reason the reaction should preferably be 7. APPLICATIONS OF SERINE completed within a short time. No loss of activ- CARBOXYPEPTIDASES ity is registered in 3 M-urea (K. BREDDAM, 7.1. Determination of C-terminal sequences unpublished results). The ability of carboxypeptidases to sequen- The rates of hydrolysis with serine carboxy- tially release amino acids from the C-terminus of peptidases are strongly affected by the nature of a peptide chain has frequently been used in the the amino acid residues in the P~ and P; positions elucidation of primary structures of peptides of peptide substrates (see Table V), and conse- and proteins. Originally metallo carboxypeptid- quently the individual steps in the sequential ases A and B which are optimally active at basic release of amino acids from peptides are charac- pH values were used (2) but recently serine terized by widely different rates. In cases where carboxypeptidases, and in particular carboxy- a slow step is followed by one or several fast steps peptidase Y, have gained wide-spread use be- the results are ambiguous and this is often cause these enzymes release all C-terminal encountered when the non-specific carboxypep- amino acids from peptides including Pro (74, tidase Y is used for the digestions. An example is 185) which is not released by the metallo carbo- shown in Figure 4, where oxidized ribonuclease xypeptidases (2). The ability of serine carboxy- is digested with carboxypeptidase Y at pH 7.0. peptidases to act on peptide amides (see section The sequence of the C-terminal segment of this 6.1) allows C-terminal sequence determinations peptide is: -Pro-Val-His-Phe-Asp-Ala-Ser-Val- on these substances although the C-terminal OH, but from the results in Figure 4 it is only amino acid residue in cases where the amino possible to deduce the correct positions of the acid amide is released faster than ammonia (23, two amino acids released first. This is because 34) has to be identified as the released amino carboxypeptidase Y catalyzed peptide hydro- acid amide. Carboxypeptidase Y functions in lysis is very slow at pH 7.0 for substrates with an denaturing agents such as 0.5% SDS (123) and 6 Asp residue in the Pt position (70). Thus, in the M-urea (72), and this allows it to be used to digest case of oxidized ribonuclease, the release of Ala

112 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM:Serine carboxypeptidases

a 8

, , [ o 20 40 6 I MINUTES 0 50 100 Figure 6. Digestion of oxidized ribonuclease with malt M I NUT E S carboxypeptidase I. Reaction conditions: 0.5 mM oxi- Figure 7. Digestion of oxidized ribonuclease with dized ribonuclease, 0.1 M-Mes, pH 5.0, 0.25 p.M-malt Ph-Hg-CPD-Y. Reaction conditions: 0.5 mM oxidized carboxypeptidase. The symbols are listed in Figure 4. ribonuclease, 0.1 M-Mes, pH 5.0, 2.2 btM-Ph-Hg-CPD- Y. The symbols are listed in Figure 4. is very slow, while the following amino acids are relased with comparatively high rates. Problems Pro-Val bond is cleaved slower than with carbo- of this nature may in some cases be met by xypeptidase Y and that Pro is not released at all. increasing the rate of the slow step or by decreas- Although a combination of the results ob- ing the rate(s) of the following fast step(s). This tained with carboxypeptidase Y and malt carbo- may be accomplished by alterations in the reac- xypeptidase I may indicate substantial parts of tion conditions, e.g. pH, ionic strength, and the sequence, a single satisfactory time course concentration of suhstrate. Thus, substrates has not been produced with either enzyme. which at the P~ position contain an Asp or Glu However, when carboxypeptidase Y modified are hydrolyzed much faster at pH 5.0 than at pH with Ph-Hg + is used a reaction course is obtained 7.0 while for those containing His at this position which easily can be interpreted (Figure 7). This the opposite relation holds. These effects of pH is because modification of carboxypeptidase Y are due to carboxypeptidase Y hydrolysing sub- with Ph-Hg ~ results in a drastic decrease in strates with charged side-chains in the P~ posi- k:=/Km for the hydrolysis of substrates with Phe tion much slower than substrates with an un- in the P~ position while the hydrolysis of sub- charged side-chain (27, 70). By lowering the pH strates with less bulky side-chains in the Pt from 7.0 to 5.0 in the digestion of oxidized position is less affected (27). Consequently, this ribonuclease Ala is released almost as fast as Ser, modification causes a much more pronounced while the His-Phe bond is cleaved with a lower decrease in the rate of cleavage of the Phe-Asp relative rate than at pH 7.0, allowingthe position bond in ribonuclease than the other peptide of Phe in the sequence to be decided (Figure 5). bonds towards the C-terminus. This experiment In cases where alterations in the reaction demonstrates that in cases where an optimiza- conditions are not sufficient to obtain an inter- tion of the conditions of the digestion experi- pretable sequential release of amino acids from ment is insufficient to produce a time course the C-terminus of peptides additional informa- which can be interpreted, modified enzymes tion may be obtained using an enzyme with a with altered specificities may be employed with different specificity. Figure 6 shows the time advantage. course for the action of malt carboxypeptidase I The extent of the digestion is also dependent on oxidized ribonuclease. It is apparent that the on the specificity of the enzyme used. This is

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 113 K. BREODAM; Serine carboxypeptidases

H-Hi s-Ser-Gl n-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Al a-Gl n-Asp-Phe-Val -Gl n-Trp-Leu-Met-Asn-Thr-OH CPD-Y: ~ ~ ~ ~ ~ ~-~ ',~-- (x--- ~-~)~---- ~-- malt-ll: (~ ~ ~-- ~ ~ ~ ~ n-- ~ ~--- ~--- ~-~ x--- n--- ~ ~--- ~-- ~T--)~--- ~---~-- ~)~ ~ ~) ~

Figure 8. Digestion of porcine glucagon with serine carboxypeptidases.Conditions: 285 itM-glucagon, 2.2 laM malt carboxypeptidase II or 1.9 p.M-carboxypeptidaseY, 0.05 M-sodium acetate, pH 4.0. The final aliquot was taken at 3 h. The arrows indicate the interpretable sequential release of amino acids from the peptide. The arrows placed in parenthesis indicate that the release of these amino acids can be measured, but their positions within this peptide segment cannot be distinguished.

demonstrated by digestion of glucagon with 7.3. Use of serine carboxypeptidases in carboxypeptidase Y and malt carboxypeptidase peptide synthesis II. The former of these enzymes will only very BERGMAN, FRUTON and FRAENKEL-CON- slowly release amino acids from peptides with RAT demonstrated in 1937 (14, 15) that chymo- Arg or Lys in the penultimate position and trypsin and papain catalyzed the condensation consequently, the Arg-Arg sequence in the of an acylamino acid and an amino acid amide middle of the glucagon peptide will represent an thus forming a peptide bond according to reac- essentially unsurmountable obstacle for this en- tion I in Scheme 7. Reactions of this type have zyme (Figure 8). However, malt carboxypepti- since been shown to exhibit equilibrium con- dase II exhibits a preference for this type of stants Ks~, of 0.3 - 0.5 M~at neutral pH values sequence (37, 38) and digests glucagon down to (46, 81, 82) and are consequently displaced in the N-terminal dipeptide, i.e. H-His-Ser-OH. favour of hydrolysis. Significant product yields Thus, the C-terminal sequence can be deter- can, however, be obtained (a) if the product mined with carboxypeptidase Y, and after ther- continuously is removed from the equilibrium, mal denaturation of this enzyme, malt carboxy- e.g. by precipitation (95, 96, 97), or (b) by using peptide II can be added to provide additional large concentrations of the reactants thereby sequence information. Recent experiments displacing the equilibrium in favour of synthesis have shown that malt carboxypeptidase II is (186), or (c) by changing the polarity of the particularly suited for the determination of C- reaction medium through the addition of orga- terminal sequences of tryptic peptides since it nic solvents (81). rapidly releases the C-terminal Lys or Arg of Protease catalyzed aminolysis reactions of such peptides and the subsequent amino acid peptide esters (reaction II, Scheme 7) are energe- residues with lower rates (K. BREDDAM and M. tically much more favourable than condensa- OTTESEN, to be published). tion reactions (reaction I) (reviewed by J. FRU- TON (56)). The ester substrate rapidly forms an acyl-enzyme intermediate which further reacts, in competition with water, with the amine nu- 7.2. Debittering of bitter peptides cleophile to form a new peptide bond. By termi- Enzymatic hydrolysates of various proteins nating the reaction before final equilibrium can frequently contain peptides with bitter taste be established through reactions where the ami- which limit their use in food. The serine carbo- nolysis product functions as substrate, e.g. via xypeptidase from wheat bran has been demon- reaction III, it is achieved that the yield of strated to release hydrophobic amino acids from aminolysis is not based on an equilibrium con- such peptides obtained by pepsin hydrolysis of stant as in reaction I but rather on the relative casein with the result that the bitterness decrea- rates of aminolysis and hydrolysis of ester sub- ses (187). This suggests that carboxypeptidases strates (see section 6.3). and other exopeptidases might have applica- While all proteolytic enzymes may catalyze tions in food chemistry. reaction I, reaction II is catalyzed only by those

114 Cadsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM: Serine carboxypeptidases

Reversal of hydrolysis

II II I II II P-NH-CH-C-O -+ +I-~-CH-C-X I P-NH-CH-C-NH-CH-C-X + H20

Aminolysis of alkyl esters

~11 O R' o II i~ oII II II P-NH-CH-C-OMe + H~I-CH-C-• P-NH-CH-C-NH-CH-C-X + MeOH

1 l~ II P-NH-CH-C-O- + +H3N-CH-C-X Scheme 7. Two different principles of enzymatic peptide synthesis.

enzymes which hydrolyse alkyl esters, i.e. serine nolysis reaction depends strongly on the particu- and sulfhydryl proteases. The structures of the lar amino acid and it is rarely higher than 50% acyl and amine components which may parti- (197). Malt carboxypeptidase I is unstable at pH cipate in the reaction are determined by the values above 7.5, and at this pH the yield of specificity of the enzyme used, and studies in this laboratory have revealed that serine carboxy- Method I peptidases, e.g. carboxypeptidase Y and the malt CPD-Y carboxypeptidases, as expected, catalyze only a Bz-X-OMe -I- H-Y-OH Bz-X-Y-OH aminolysis reactions with amino acids and amino acid derivatives as amine components b Bz-X-Y-OH P Bz-X-Y-OMe (33, 38, 196, 197) as opposed to endopeptidases Method II that in addition to amino acid derivatives also CPD-Y Bz-X-OMe -t- H-Y-OMe Bz-X-Y-OMe accept polypeptides as nucleophiles (136, 149).

Consequently, when a carboxypeptidase is used Method III a peptide chain will only be elongated by a single CPD-Y a Bz-X-OMe -t- H-Y-NH 2 D Bz-X-Y-NH 2 amino acid residue in each aminolysis reaction. CPD-Y This may be achieved in three different ways b Bz-X-Y-NH 2 = Bz-X-Y-OH using amino acids, amino acid amides, or amino acid methyl esters as amine components accord- c Bz-X-Y-OH . D Bz-X-Y-OMe ing to the reactions listed in Scheme 8. Scheme 8. Three different ways of performing one When amino acids are used as nucleophiles elongation step in carboxypeptidase Y catalyzed pep- (method I, reaction a) it is important to perform tide synthesis. Bz-X~-OMerepresents the peptide ester the reaction at a pH where the enzyme exhibits functioning as acyl component in the reaction, and high esterase activity and almost no peptidase H-Y-OH, H-Y-OMe, and H-Y-NH2 represent the amino acid, amino acid methyl ester, and amino acid activity such that the product formed in the amide, respectively,functioning as amine components reaction is not degraded by the enzyme. In the in the reactions. Bz-X-Y-~OMe represents the acyl- case ofcarboxypeptidase Y this is achieved at pH component in the subsequent elongation reaction. The > 9 (see section 5.2) where the enzyme remains esterification reactions lb and Illc are performed stable (27). Unfortunately, the yield of the ami- chemically.

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 115 K. BREDDAM"Serine carboxypeptidases aminolysis using amino acids as nucleophiles (29). These results demonstrate that the side- was below 10% conceivably due to an unfavou- chain specificity of the enzyme is very important rable ratio of esterase to peptidase activity (K. for the yield. Consequently, it would be advanta- BREDDAM, unpublished results). However, malt geous to have several serine carboxypeptidases carboxypeptidase II is more stable at basic pH with different specificities available. This is illus- values and at pH 8.5 the yields of aminolysis trated by Bz-Arg-Ala-OMe being produced in appear to be of the same magnitude as obtained 50% yield from Bz-Arg-~OBu and H-AIa-OMe with carboxypeptidase Y (38). using malt carboxypeptidase I compared to 2% The use of amino acid esters as amine compo- with carboxypeptidase Y which hydrolyses Bz- nents (Scheme 8, method II) is complicated by Arg very slowly (K. BREDDAM, unpub- the fact that the aminolysis product, like the acyl lished results). component used in the reaction, is a peptide Since carboxypeptidase Y and malt carboxy- ester and hence, it may react further in one or peptidase II are the only serine carboxypeptid- more additional steps resulting in lower yields of ases which are available in such quantities that the desired products (196). However, with car- they can be commercially utilized in enzymatic boxypeptidase Y it has been demonstrated that synthesis, it is fortunate that the specificity of the yield of the initial aminolysis product can be carboxypeptidase Y with respect to the P, posi- drastically improved by utilizing that N-blocked tion of the ester substrate may be altered by amino acid benzyl esters are much better sub- modification of its sulfhydryl group with mer- strates of carboxypeptidase Y than the corre- curials (see section 6.4.1). Thus, work in this sponding methyl esters (29). Thus, by using laboratory has shown that carboxypeptidase Y N-blocked amino acid benzyl esters as acyl-com- modified by Ph-Hg + is particularly suitable for ponents and amino acid methyl esters as amine the synthesis of peptide esters Bz-X-Y-OMe components, peptide methyl esters are formed where X is a nonbulky amino acid residue and Y as the major product because they are turned is a bulky amino acid residue. This is because the over by the enzyme with a lower rate than the preference of this enzyme towards ester sub- initial benzyl ester acyl component. Similarly, it strates with bulky amino acid residues in the P, has been shown that high yields of the initial position is lower than that observed with the aminolysis product can be obtained in cases unmodified enzyme such that the reactivity of where the side-chain specificity of the enzyme is Bz-X-Y relative to Bz-X (-OBzl) is such that the peptide ester formed as aminolysis reduced when compared with the unmodified product is a poor substrate of carboxypeptidase enzyme. The HPLC chromatograms in Figure 9 Y relative to the substrate used as acyl compo- compare the reactant composition ofa carboxy- nent in the reaction (29). Thus, when e.g. Bz- peptidase Y catalyzed synthesis of Bz-Ala-Leu- Ala functions as acyl component and OMe from Bz-Ala and H-Leu-OMe (panel H-Gly-OMe as amine component, the amino- A) with the corresponding synthesis with carbo- lysis product, Bz-Ala-Gly-$OMe is a poor sub- xypeptidase Y modified with Ph-Hg * (panel B). strate of carboxypeptidase Y relative to Bz-Ala-$ It is apparent that the initial coupling product, OMe, and consequently, Bz-AIa-Giy~OMe re- Bz-Ala-Leu-OMe, is accumulated in much acts no further at the concentrations of enzyme higher yield with the modified enzyme than with and time needed to consume all Bz-AIa the unmodified enzyme. This is due to the ability of carboxypeptidase Y When amino acid amides are used as nucleo- to hydrolyse ester substrates with Ala in the P, philes (Scheme 8, method Ill, reaction a) it is not position much faster than those with Gly in this critical to perform the reaction at high pH since position (27). Conversely, when H-Phe-OMe is the esterase activities ofcarboxypeptidase Y and used as the nucleophile the product, Bz-Ala- the malt carboxypeptidases are far higher than Phe-~OMe, is a better substrate than Bz-Ala-$ their amidase activities (28, 37) regardless ofpH OMe, because Phe is preferred to Ala in the P~ such that the product, a peptide amide, usually position (27), and Bz-Ala-Phe~OMe reacts fur- is not degraded by the enzyme. The yield of ther so that it is obtained in a yield of 2% only aminolysis is 85 - 95% for most amino acid

116 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K BREDDAM"Serine carboxypeptidases

X-A-B-OH

NH~I k~ H-B-OH X-A-B-NH 2 III

4 H-B-N E IH~ c 3 8 X-A-OH 113 5 10 N Scheme 9. Carboxypeptidase Y catalyzed deamidation reactions. Reactions I, II, and IIIrepresent amidase, tg peptidyl amino acid amide hydrolase, and peptidase

Z activities, respectively. X = N-blocking group; A and B In = amino acid residues. er- 0 m

! quently of limited value in the deamidation of polypeptide amides. The serine carboxypeptidases also exhibit amidase activity (Scheme 9, 5 reaction I) and in the case ofcarboxypeptidase Y the formed peptide is not degraded via the peptidase activity of the enzyme (Scheme 9, -4 , , , J , i i i i i i , t , , reaction III) provided that the reaction is per- 0 2 4 6 8 10 12 14 formed at pH l0 (23). However, carboxypeptid- TIME (MINUTES) ase Y in addition exhibits peptidyl amino acid Figure 9. HPLC chromatograms ofpeptides formed by amide hydrolase activity and consequently also CPD-Y (Panel A) and Ph-Hg-CPD-Y (Panel B) cata- releases the C-terminal amino acid amide from lyzed reaction of Bz-AIa-OBzl in the presence of peptide amides (Scheme 9, reaction II). The H-Leu-OMe. The following peaks were assigned: 1: relative rates of reactions I and II are strongly Bz-Ala-OH (hydrolysis product), 2: Bz-AIa-Leu-OH, dependent on the C-terminal sequence of the 3: Bz-Ala-Leu-OMe (aminolysis product), 5: Bz-Ala- peptide amide (Table IX) but usually the yield of OBzl (substrate), 4, 6, 7, 8, 9, 10 are oligomerization the deamidated product formed via reaction I is products containing more than one leucyl residue. The lower than 70% (23, 34). Thus, deamidation results are from ref. 29. with carboxypeptidase Y can generally not be performed in a satisfactory yield but, fortunately, it has recently been demonstrated in this laboratory that most peptide amides can be amides using either the malt carboxypeptidases deamidated in significantly higher yields (80- or carboxypeptidase Y, but further elongation of 100%) using carboxypeptidase Y modified with the peptide amide produced in reaction IIIa phenacylbromide (Table IX). This difference requires that it is deamidated (reaction IIIb) between the two enzymes is due to alkylation of prior to esterification (reaction IIIc). Chemical a methionyl residue in the S] binding site of methods are not suitable for the deamidation carboxypeptidase Y enhancing the amidase ac- reaction and no native enzyme has been de- tivity, i.e. reaction I in Scheme 9, while the scribed which specifically releases NH3 from peptidyl amino acid amide hydrolase activity, peptide amides. Serine and sulfhydryl proteases i.e. reaction II in Scheme 9, is decreased (see do exhibit amidase activity, but in addition they section 6.4.2). The amidase and peptidyl amino cleave internal peptide bonds, and are conse- acid amide hydrolase activities of malt carboxy-

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 117 K. BREDDAM"Serine carboxypeptidases peptidase I are influenced in a similar manner by Table IX. Deamidation of peptide amides by carboxy- addition of phenylguanidine to the reactiofi peptidase Y and carboxypeptidase Y modified with medium (see section 6.2) and when used in malt phenaeylbromide. carboxypeptidase I catalyzed deamidation reac- Peptide amide % Yield tions this substance increases the yields of dea- CPD-Y PAB-CPD-Y midation substantially (30). Although the unique side-chain specificity of malt carboxy- Z-Ala-Gly-NH2 2 10 peptidase I (30) potentially could be useful in Z-Val-GIy-NH~ 46 49 deamidation reactions, viz. deamidation of pep- Z-AIa-Ser-NH2 0 35 tide amides with C-terminal lysyl, arginyl or Bz-Phe-GIn-NH2 11 81 histidyl residues, malt carboxypeptidase I is Bz-Ala-AIa-NHz 31 100 available only in small quantities and conse- Z-Gly-AIa-NH2 IO0 Z-Val-Ala-NH2 94 quently, deamidation of most peptide amides is Bz-Ala-Thr-NH2 9 85 best performed with carboxypeptidase Y mo- Bz-Ala-VaI-NH., 10 96 dified with phenacylbromide. Bz-AIa-IIe-NH2 10 92 It is thus apparent that peptide chains may be Bz-AIa-Leu-NH2 60 98 elongated by reactions catalyzed by serine carbo- Bz-AIa-Phe-NH2 66 96 xypeptidases, provided the peptide ester acyl- Bz-Phe-His-NH2 28 92 component is a substrate of the enzyme. Unfor- Z-AIa-Met-NH_, 36 100 tunately, Pro cannot be incorporated by either of The results are from ref. 34. the methods I, II, and III in Scheme 8, and Asp and Glu only when the carboxylic acid group in the side-chain is blocked, e.g. by esterification (196, 197). However, replacements of specific amino acid residues in the S~ binding site by therefore be used in step-wise synthesis in com- site-directed mutagenesis can conceivably be bination with serine carboxypeptidases. How- employed to enhance the binding of such proble- ever, presumably they will primarily be used to matic nucleophiles. The carboxypeptidase Y catalyze reactions in which peptides function as containing Leu at position 398 in place of Met amine components. In general, enzyme assisted exhibits an increased preference for hydropho- peptide synthesis is characterized by somewhat bic residues in the P~ position and this derivative lower yields than those characteristic of the would presumably bind hydrophobic nucleo- chemical methods (61). Nevertheless, enzyma- philes better than the unmodified enzyme. In tic peptide synthesis have advantages in many analogy, replacements with hydrophilic amino cases since it is stereospecific and requires min- acid residues might favour binding of hydrophi- imal protection of functional groups in the lic nucleophiles. No general rule can be formu- side-chains (198). lated to decide which of the methods in Scheme In conventional peptide synthesis ester groups 8 is the most suitable for incorporation of a can be used to protect carboxylic acid groups but specific amino acid, but the recent development this requires that the ester group subsequently is which allows the deamidation ofpeptide amides removed without otherwise affecting the in high yields suggest that method III in spite of elongated peptide. ROYER (160) has previously the extra reaction step (reaction IIIb) will gain demonstrated that the high esterase and low more wide-spread use, especially when it is taken peptidase activity ofcarboxypeptidase Y at pH into consideration that the yields obtained by > 8 renders this enzyme a useful tool in such methods I and II rarely exceed 65% which is reactions. However, observ/ttions in this labora- significantly less than the expected overall yield tory suggest that carboxypeptidase Y even under of method III (80 - 90%). these conditions exhibits significant peptidase Serine and sulfhydryl endopeptidases do not activity, in particular towards peptides with catalyze reaction I in Scheme 8 but they do hydrophobic amino acid residues in the P~ and P~ catalyze reactions II and IIIa (136, 149) and may positions. Thus, after removal of the ester group

118 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDOAM:Serine carboxypeptidases

I. Conversion of peptides to peptide esters tO0 (a) Bz-X-Y-OH + NeOH ~ Bz-X-OMe + H-Y-OH

(b) Bz-X-Y-OH + H-Z-OMe ~ Bz-X-Z-OMe + H-Y-OH

If. Conversion of peptldes to peptide amides

(a) Bz-X-Y-OH + NH3 Bz-X-NH2 + H-Y-OH ~ 50 (b) Bz-X-Y-OH + H-Z-NH2 Bz-X-Z-NH2 + H-Y-OH

Ill. Conversion of peptides to other peptldes

Bz-X-Y-OH + H-Z-OH ) Bz-X-Z-OH + H-Y-OH Scheme l 0. CarboxypeptidaseY catalyzedexchange of C-terminal residues in peptides. X, Y, and Z represent amino acid residues. I 2 3 4- REACTION TIME (HOURS) Figure 10. The action of carboxypepfidase Y on Bz- Lys-Ala-OH in the presence of H-Thr-NH2. Bz-Lys- from the peptide ester carboxypeptidase Y may Ala, -O-O-; Bz-Lys-OH, -A-&-; Bz-Lys-Thr-NH2, degrade the unblocked peptide. Carboxypeptid- -m-m-; Bz-Lys-Ala-Thr-NH2, -x-x-; Bz-Lys-Thr- ase Y modified with phenacylbromide has been Thr-NH2 + Bz-Lys-Thr-Thr-OH,l O i O - . The results shown to exhibit negligible peptidase activity are from ref. 31. (see section 6.4.2) and compared with unmodified carboxypeptidase Y this modified enzyme removes ester groups from peptide esters more specifically and can consequently with advan- that other products could be formed as well in tage be applied in such reactions (34). cases where the acyl component is a poor substrate of the enzyme (30). The product composition (Figure 10) indicates that Bz-Lys-~Ala-OH 7.4. Exchange of C-terminal amino acid functions as acyl component in three reactions residues in peptides forming Bz-Lys-Thr-NH2, Bz-Lys-Ala-Thr- All hydrolytic reactions catalyzed by carboxy- NH2, and Bz-Lys-OH, and that Bz-Lys-Thr peptidase Y proceed via an acyl-enzyme inter- reacts further to yield Bz-Lys-Thr-OH and Bz- mediate, and studies in this laboratory have Lys-Thr-Thr-NH2 (Scheme 11). The formation shown that all substrates of the enzyme in of these additional products is due to Bz-Lys principle can be used as acyl-components in Ala-OH being a very poor substrate of carboxy- transacylation reactions to other nucleophiles peptidase Y (27) such that the rates of the than water. It is of particular interest that un- otherwise fast reactions I and III are reduced to blocked peptides may function as acyl-donors approximately the rates of reactions II, IV and V. such that the C-terminal amino acid residue can The lysyl residue in the P~ position of the be exchanged for various other groups as shown substrate is unquestionably the cause for this in Scheme 10 (23, 25). atypical reaction course. Consistent with this the Carboxypeptidase Y catalyzed transpeptida- reaction course was drastically altered when tion reactions using N-blocked dipeptides as acyl carboxypeptidase Y modified by Hg t+ was used, components and amino acid amides as amine hence increasing the preference for Lys in the P, components normally result in formation of position (26) (Figure 11): only Bz-Lys-Thr-NH2 only two products, a transpeptidation product (95%) and Bz-Lys-OH (5%) were formed. With where the C-terminal amino acid residue has malt carboxypeptidase II which exhibits high been replaced and a hydrolysis product. How- activity towards Bz-Lys-AIa-OH a yield of 85% ever, a carboxypeptidase Y catalyzed reaction was obtained using only 0.5 ~tM enzyme as with Bz-LyskAla-OH as acyl component and compared with 12 btM modified carboxypeptid- H-Thr-NH2 as amine component demonstrated ase Y. These results demonstrate that the speci-

Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 119 K. BREDDAM"Serine carboxypeptidases

I. Reactions with Bz-Lys-Ala-OH:

(a) Bz-Lys-Ala-OH + H-Thr-NH2 > Bz-Lys-Thr-NH2 + H-AIa-OH (transpeptidation)

(b) Bz-Lys-Ala-OH + H-Thr-NH2 Bz-Lys-Ala-Thr-NH2 + H20 (condensation)

II. Reactions with Bz-Lys-Thr-NH2

(a) Bz-Lys-Thr-NH2 + H20 Bz-Lys-Thr-OH + NH3 (hydrolysis)

(b) Bz-Lys-Thr-NH2 + H-Thr-NH2 > Bz-Lys-Thr-Thr-NH2 + NH3 (transpeptidation) Scheme 11. Reaction courses for the action of carboxypeptidase Y on Bz-Lys-Ala-OH in the presence of a nucleophile (H-Thr-NH2).

specificity of the enzyme employed imposes amino acid residues in peptides, incorporation severe restrictions on the type of reactions which of labelled amino acids as well as reporter groups are feasible and in addition, they demonstrate and conversion of peptides to peptide esters are how chemically modified derivatives of en- possibilities to be considered. zymes may be useful. Porcine and human insulins only differ at the Although the yields of carboxypeptidase Y C-terminal position of the B-chain, porcine catalyzed transpeptidation reactions are strong- insulin containing an alanyl residue and human ly dependent on the structures of both the acyl insulin a threonyl residue. The semisynthetic and amine components used in the reaction (23, conversion of porcine insulin to human insulin 24) they represent attractive possibilities in pro- has been solved in various ways (26, 32, 58, 94, tein semisynthesis. Exchange of C-terminal 99, 137, 138). In the procedure originating from this laboratory (26) the C-terminal amino acid residue of porcine insulin is exchanged by a carboxypeptidase Y catalyzed transpeptidation reaction using porcine insulin as acyl component and H-Thr-NH2 as amine component 100 forming human insulin amide, des(Ala)B3~ - NH2)B3~ However, like Bz-Lys porcine insulin is a poor substrate of carboxy-

Of) peptidase Y because it too contains a lysyl I-- residue at the P, position. The reaction course of Z ,:t 50 the transpeptidation reactions with porcine in- I- t..) sulin and Bz-Lys-Ala-OH are consequently very I.U reminiscent in terms of products formed and the n,- r rather low yields of the transpeptidation products. Fortunately, however, the yield of human insulin amide could be increased from approxi- ! mately 25% to approximately 65% by modifica- 1 2 5 tion of carboxypeptidase Y with H~ ++ in analogy TIME (HOURS) with the observations with Bz-Lys-*Ala-OH, and Fig. 11. The action of CI-Hg-CPD-Y on Bz-Lys-Ala- human insulin could subsequently be obtained OH in the presence of H-Thr-NH2. Bz-Lys-AIa-OH, from human insulin amide by a carboxypeptid- -O-e-; Bz-Lys-OH, -169 Bz-Lys-Thr-NH2, ase Y catalyzed deamidation reaction in essen- -B-m-. The results are from ref. 31. tially quantitative yield (32).

120 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986 K. BREDDAM: Serine carboxypeptidases

ACKNOWLEDGEMENTS and anilide substrates and the effects of some I am indebted to Professor MARTIN OTTESEN environmental factors. J. Biochem. (Tokyo) 78, for his many suggestions, continued support 617-626 (1975) during the work, and for revising the ma- 9. BAMFORTH, C. W., H. L. MARTIN & T. WAIN- WRIGHT: A role for carboxypeptidase in the nuscript. Thanks are also due to Professors solubilization of barley 13-glucan. J. Inst. Brew. BENT FOLTMANN and SVEND OLAV ANDERSEN 85, 334-338 (1979) for valuable suggestions to improve the ma- 10. BAUDYS,M., V. KOSTKA,G. HAUSDORF,S. FITT- nuscript. I also want to thank Drs. JACK JOHAN- KAU & W. E. HOHNE: Amino acid sequence of the SEN, F. WIDMER, IB SVENDSEN, STEEN BECH tryptic SH-peptide ofthermitase, a thermostable SORENSEN, JAKOB WINTHER, LENE BECH and serine proteinase from thermoactinomyces vul- MORTEN KIELLAND-BRANDT for cooperation gaffs. Int. J. Peptide Res. 22, 66-72 (1983) on parts of the project. Finally, the excellent 11. BAXTER,E.D.: Purification and properties of technical assistance of Mss IRENE SIMONSEN, malt carboxypeptidases attacking hordein. J. PIA BREDDAM, MERETE SONNE and Mr. Inst. Brew. 84, 271-275 (1978) 12. BENDER, M. L. & F. J. KEZDY: Mechanism of THORKILD BEENFELDT iS acknowledged. action of proteolytic enzymes. Ann. Rev. Bio- chem. 34, 49-76 (1964) 13. BENDER,M. L., G. E. CLEMENT,C. R. GUNTER & F. J. KEZDY: The kinetics of chymotrypsin reac- REFERENCES tions in the presence of added nucleophiles. J. 1. AFROZ, H., K. OTTO,R. MI~/LLER& P. FUHGE: On Am. Chem. Soc. 86, 3697-3703 (1964) the specificity of bovine spleen cathepsin B2. 14. BERGMANN,M. & J. S. FRUTON: Some synthetic Biochim. Biophys. Acta 452, 503-509 (1976) and hydrolytic experiments with chymotrypsin. 2. AMBLER, R. P.:CarboxypeptidasesA and B.Eds. J. Biol. Chem. 124, 321-329 (1938) C. H. N. Hirs & S. N. Timasheff, Academic Press. 15. BERGMANN,M. & H. FRAENKEL-CONRAT:The Methods Enzymol. 25,262-272 (1972) role of specificity in the enzymatic synthesis of 3. AULD, D. S. & B. L. VALLEE: Kinetics of carboxy- proteins. J. Biol. Chem. 119, 707-720 (1937) peptidase A. The pH dependence of tripeptide 16. BIEDERMANN, K., U. MONTALI, B. MARTIN, I. hydrolysis catalyzed by zinc, cobalt and manga- SVENDSEN& M. OTTESEN: The amino acid sequ- nese enzymes. Biochemistry 9, 4352-4359 (1970) ence of proteinase A inhibitor 3 from baker's 4. BACHOVCHIN,W. W. & J. D. ROBERTS:Nitrogen- yeast. Carlsberg Res. Commun. 45, 225-235 15 nuclear magnetic resonance spectroscopy. (1980) The slate of histidine in the catalytic triad of 17. BIRKTOFT,J. J., D. M. MATTHEWS,T. A. POULUS tt-lytic protease. Implications for the charge-relay & J. KRAUT: A crystallographic view of the serine mechanism of peptide-bond cleavage by serine protease mechanism. Abstract: V. Linderstrom- proteases. J. Am. Chem. Soc. 100, 8041-8047 Lang Conference. Vingsted, Denmark (1975) (1978) 18. BLOBEL,G. & B. DOBBERSTEIN:Transfer of pro- 5. BACHOVCHIN,W., R. KAISER,J. H. RICHARDS& J. teins across membranes. J. Cell. Biol. 67,835-851 D ROBERTS:Catalytic mechanism ofserine pro- (1975) teases: Reexamination of the pH dependence of 19. BLOW, D. M., J. J. BIRKTOFT & B. S. HARTLEY: the histidyl ~J~3-c-2-~ coupling constant in the Role of a buried acid group in the mechanism of catalytic triad of tt-lytic protease. Proc. Natl. chymotrypsin. Nature 221,337-340 (1969) Acad. Sci. USA 78, 7323-7326 (1981) 20. BLUMBERG,S & B. L. VALLEE:Superactivation of 6. BAI, Y. & R. HAVASHI: Properties of the single thermolysin by acylation with amino acid N-hy- sulfhydryl group of carboxypeptidase Y. Effects droxysuccinimide esters. Biochemistry 14, 2410- of alkyl and aromatic mercurials on activities 2419 (1975) toward various synthetic substrates. J. Biol. 21. BREDDAM,K.: Isolation and characterization of Chem. 254, 8473-8479 (1979) carboxypeptidase from yeast. Thesis, in Danish, 7. BAL Y., R. HAYASHI& T. HATA: Kinetic studies of University of Copenhagen, september (1975) carboxypeptidase Y. II. Effects of substrate and 22. BREDDAM,K., T. J. BAZZONE,B. HOLMQUIST&B. product analogs on peptidase and esterase activi- L. VALLEE: Carboxypeptidase of S. griseus. Im- ties. J. Biochem. (Tokyo) 77, 81-88 (1975) plications of its characteristics. Biochemistry 18, 8. BAI,Y., R. HAYASHI& T. HATA: Kinetic studies of 1563-1570 (1979) carboxypeptidase Y. III. Action on ester, amide, 23. BREDDAM, K., F. WIDMER & J. T. JOHANSEN:

Cadsberg Res. Commun. Vol. 51, p. 83-128, 1986 121 K. BREDDAM" Serine carboxypeptidases

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Accepted by S. O. ANDERSEN

128 Carlsberg Res. Commun. Vol. 51, p. 83-128, 1986