Comparative studies on human carboxypeptidases B and N

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 197, No. 2, October 15, pp. 487-492, 1979

Comparative THOMAS Division

Studies

J. McKAY,~ of Laboratories

ARTHUR and

on Human

Carboxypeptidases

W. PHELAN,

Research, New York

State

AND THOMAS Department

of Health,

B and N1 H. PLUMMER, Albany, New York,

JR.~ 12201

Received February 2, 19’79; revised June 14, 1979 A series of dicarboxylic acid bi-product analogs of lysine and arginine have been tested as competitive inhibitors of human pancreatic carboxypeptidase B and human plasma carboxypeptidase N. The most effective derivative was guanidinoethylmercaptosuccinic acid with Kis of 0.5 and 1.0 x 10m6M for carboxypeptidases B and N, respectively. Values for the all-carbon guanidinopropylsuccinic acid were similar. In addition the kinetic parameters, K, and k,,,/K,, have been determined for the hydrolysis of benzoyl-alanyl-lysine and benzoylalanyl-arginine by human carboxypeptidases B and N. These substrates have been proposed for use in improved spectrophotometric assays. An enhanced affinity of these substrates versus benzoyl-glycyl-lysine or benzoyl-glycyl-arginine indicates a significant participation of the penultimate amino acid in catalysis of substrate.

Enzymes that release the basic amino acids, lysine and arginine, from the COOHterminal position of peptides and proteins have been known since the first report by Waldschmidt-Leitz et al. (1). Studies involving human carboxypeptidase B-type enzymes from different tissues are important because of possible physiological roles for these enzymes. Human pancreatic carboxypeptidase B (2, 3) is a zinc metalloenzyme of approximately M, = 34,000 that may function not only as a digestive enzyme, but also as the final step in the conversion of proinsulin to insulin (4). Human plasma carboxypeptidase N is a zinc metalloenzyme of approximately M, = 270,000 that contains 17% carbohydrate (5). This enzyme inactivates circulating physiologically active peptides, such as bradykinin (6) and anaphylatoxins C3a and C5a (‘7), by releasing COOHterminal arginine. McKay and Plummer (8) synthesized a series of dicarboxylic acid sulfur-containing bi-product analogs of lysine and arginine ‘This work was supported in part by National Institutes of Health Research Grant GM-11764, awarded by the National Institute of General Medical Sciences, PHSDHEW. *Present address: HFF-144, Food and Drug Administration, Washington, D. C. 20204. 3To whom all correspondence should be directed.

that are the most effective competitive inhibitors known for bovine carboxypeptidase B. This paper compares the effects of similar bi-product analogs as inhibitors of human pancreatic carboxypeptidase B and plasma carboxypeptidase N. It will also detail the kinetic parameters and propose Bz-Ala-Arg4 and Bz-Ala-Lys as improved spectrophotometric substrates for these enzymes. MATERIALS

AND METHODS

DL-Benzylsuccinic acid (Burdick and Jackson), benzoyl-lalanine, benzoyl-L-valine (Bachem), isobutylchloroformate, 1,1,2-tricarbethoxyethane, acrylonitrile (Aldrich), arginine-HCl, hippuryl-L-lysine (Sigma), hippuryl-L-arginine (Peninsula), and N-e-tBoc-Llysine (Chemical Dynamics) were used as supplied. N-Methylmorpholine (Aldrich) was redistilled and stored under nitrogen at - 12°C. Hippuryl-L-argininic acid was purchased (Vega-Fox) or was synthesized (9) and was chromatographed on a 7.6 x 67-cm column of Sephadex G-25 equilibrated in 0.1 N acetic acid before use. The bi-product competitive inhibitors

“Abbreviations used: AE, aminoethyl; AP, aminopropyl; t-Boc, t-butyloxycarbonyl; Bz, benzoyl; BzlSA, benzylsuccinic acid; GE, guanidinoethyl; GP, guanidinopropyl; MSA, mercaptosuccinic acid; SA, succinic acid; THF, tetrahydrofuran; Z, benzyloxycarbonyl. Amino acid symbols denote the L configuration unless noted otherwise. 487 0003-9861/79/120487-06$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

488

MCKAY,

PHELAN,

GEMSA, GPMSA, AEMSA, and APMSA were synthesized by the method of McKay and Plummer (8).

Carboxypeptidases Human plasma carboxypeptidase N was prepared from pooled, outdated human plasma by the method of Plummer and Hurwitz (5). Human pancreatic carboxypeptidase B was prepared from the acetone powders of pancreata kindly furnished by K. Amiraian of this Division and later by E. P. Lazzari (The University of Texas at Houston). Our method of isolation was similar to that of Brodericket al. (2) with the exception that affinity chromatography on LLeu-DArg-agarose (10) was substituted for their final step of Sephadex G-100 chromatography. Bovine pancreatic carboxypeptidase B was obtained from pancreatic juice (11). Absorbance indices, E:?~ (280 nm), were bovine and human pancreatic carboxypeptidase B, 21 (12) and human plasma carboxypeptidase N, 11.9 (5). The latter enzyme was also quantitated by the method of Lowry et al. (13) using crystalline bovine serum albumin as the standard.

Enzyme

and Znhibitor

Assays

Enzymatic activity was determined spectrophotometrically at 254 nm with either Bz-Gly- or Bz-Ala- substrates. The assay was performed at 37°C in 0.05 M Tris-HCl buffer, pH 7.6, containing 0.5 M NaCl by the procedure of Wolff et al. (14) as modified by Kycia et al. (15). The constants, K, and k,,,, were calculated from data according to the method of Lineweaver and Burk (l/v vs l/S) (16) when K, > 0.6 and the method of Hanes-Woolf (S/V vs S) (17) when K, < 0.6. Substrate concentrations were varied from 0.2 to 1.0 mM for all assays except that of human pancreatic carboxypeptidase B and Bz-Gly-Lys. Substrate concentrations to determine this latter K, were 6.2 to 90 mM, and assays were performed on a Radiometer pH stat with TTT 60 titrator in a lo-ml thermostated reaction vessel at 37°C with 25 mM NaOH as a titrant. Enzyme concentrations varied with the substrate and ranged from 1.1 x 10e7 to 1.1 x lOA” M. Inhibitor binding constants were determined graphically by Dixon plots (18) using three concentrations of substrate (0.3, 0.5, and 1.0 mM) and four or five concentrations of inhibitor in duplicate. Linear representation of data was determined by regression analysis. The final concentrations of enzyme were 1.44 and 1.78 x 10m8M for human carboxypeptidases B and N, respectively, and 7.6 X 10mRM for bovine carboxypeptidase B.

Synthetic

Procedures

(A) Preparation

N-e-t-Boc-Lys

of Bz-Ala-substrates. Arg-HCl or were coupled with Bz-Ala by isobutyl-

AND PLUMMER chloroformate in THF at -12°C by the method of Izumiya et al. (19). The pH of resultant reaction mixtures was lowered to 2.5 with 2 N HCl and excess solvent was removed by flash evaporation. The .s-t-Boc was released from lysine by 4 N HCl in ethylacetate (20). The residual oil of the arginyl derivative was dissolved in 0.2 M pyridine formate, pH 2.34, and applied to a 2.5 x 40-cm column of Dowex 50-X8 (Aminex Q150S, Bio-Rad) equilibrated in 0.6 M pyridine formate, pH 5.28. Elution was performed by equilibration buffer at 50°C at a flow rate of 39.6 ml crne2 h-’ and fractions of 25 ml were collected. Product was detected by Sakaguchi reaction of suitable aliquots (21). The oil from the lysine derivative was dissolved in 0.2 M pyridine formate, pH 2.35, and applied to the aforementioned Dowex column equilibrated in 0.2 M pyridine formate, pH 3.24. After sample addition, elution was accomplished by a gradient using a constant volume elution reservoir of equilibration buffer (1000 ml capacity) and 0.4 M pyridine formate, pH 5.28. Product was detected by ninhydrin reaction of suitable aliquots (22). Pertinent fractions were pooled, lyophilized, and dried in a vacuum at 100°C prior to elemental analysis. Anal. Calcd for benzoyl-alanyl-clysine-3/4 H20: C, 57.40; H, 7.32; N, 12.56. Found: C, 57.57; H, 7.27; N, 12.51. Anal. Calcd for benzoyl-alanyl-L-arginine-l/2 H,O: C, 53.63; H, 6.70; N, 19.55. Found: C, 53.57; H, 6.68; N, 19.36. Yields varied from 35 to 49% for different preparations. The materials yielded 1:l ratios of alanine to lysine or arginine after acid hydrolysis and amino acid analysis. However, enzymic digestion with carboxypeptidases B or N indicated incomplete hydrolysis of substrate. Since racemization of alanine was possible during synthesis, both derivatives were resubmitted to chromatography on a 2.5 x 45-cm column of Dowex 50-X8 equilibrated in 0.2 M pyridine formate, pH 4.25, for Bz-Ala-Lys or in 0.4 M pyridine formate, pH 4.25, for Bz-Ala-Arg. Elution was performed in equilibration buffers under aforementioned conditions. Each preparation separated into two minor and one major, most retarded peak. Incubation of aliquots from each peak with human carboxypeptidase N and analysis of the digestion mixture on the amino acid analyzer revealed that only the major peak would hydrolyze completely. The other two peaks from either Bz-Ala-Lys or Bz-Ala-Arg showed little enzymic hydrolysis and were not studied further. This step reduced final product yield by nearly 50%. (B) GPSA. To a solution of sodium (100 mg) in anhydrous ethanol (20 ml) was added 1,1,2-tricarbethoxyethane (53.7 g) and acrylonitrile (12.8 g). The resulting reaction was incubated at 65°C for 4 h and then allowed to proceed overnight at room temperature. The oily 2-cyanoethyl-1,1,2-tricarbethoxyethane (40 ml) was removed by distillation (bp 185 to 195”C,

HUMAN

B AND N: COMPARATIVE

CARBOXYPEPTIDASES

reflecting a greater efficiency of hydrolysis with the latter. Furthermore, all carboxypeptidases show a pronounced improvement in specificity when penultimate alanine is substituted for glycine, as evidenced by significantly lower K, values and higher k&K,,, values. Assays of carboxypeptidase N with any of the substrates in Table I show less linearity than the pancreatic carboxypeptidases, apparently due to product inhibition from lysine and arginine (see data in Table II).

2.5 mm Hg). The distillate was mixed with water (2 ml), ethanol (120 ml), acetic acid (50 ml), and platinum oxide (200 mg) and shaken in a hydrogen atmosphere (50 psi) for 48 h. The ir spectrum showed only a trace of nitrile (2240 cm-‘). After removal of the catalyst by filtration, 6 N HCI (250 ml) was added to the filtrate. This mixture was heated on a steam bath overnight during which time most of the ethanol evaporated. The crude aminopropylsuccinic acid was isolated by ion-exchange chromatography as previously described for AEMSA (8). Guanidination with 2-guanyl-3,5-dimethylpyrazole was carried out as previously described for GEMSA (8) and the reaction mixture was chromatographed in the same manner. The yield was 15 g, 32%. Recrystallization from 80% methanol/water gave analytically pure material, mp 204 to 206°C. Anal. Calcd for GPSA: C, 44.23; H, 6.96; N, 19.34. Found: C, 44.20; H, 7.01; N, 19.26.

Competitive Inhibitors

Since the dicarboxylic acid bi-product analogs of lysine and arginine were the most efficient inhibitors of bovine carboxypeptidase B (8) these were tested on the human enzymes. The results are listed in Table II. Guanidinopropylsuccinic acid (GPSA; see Fig. 1) is the all-carbon analog of the very efficient GEMSA (8). Dixon plots (Fig. 2) were typical of competitive inhibition for both human pancreatic carboxypeptidase B and human plasma carboxypeptidase N. Competitive kinetics were confirmed by replots of slope vs l/S (not shown) and by Cornish-Bowden plots (25) (Fig. 2, inset) which gave parallel lines. The effect of replacing a P-methylene group with a sulfur, as was present in all of our original inhibitors (8), was negligible as Ki values

RESULTS

Kinetic Data

The data given in Table I illustrate that pancreatic carboxypeptidases have a much greater affinity for side chain guanidino groups than side chain amino groups. This preference results in decidedly lower K, values with arginyl substrates. The difference is less pronounced with plasma carboxypeptidase N. While the K, values with this enzyme are somewhat lower for both arginyl substrates, the value for k,d K, is greater for both lysyl substrates, TABLE KINETIC

CONSTANTS

Human carboxypeptidase

I

FOR CARBOXYPEPTIDASES

Human carboxypeptidase

N

Bovine Carboxypeptidase

Km (mm

km, (s-7

kea,lKm

Km (mm

km, (s-‘1

k,atlKm

Km (mM)

kc,, (s-‘1

90” 0.24” 1.1 0.086

170 230 878 426

1.9 960 798 4953

1.4b 0.65d 0.35 0.28

16 4 352 139

11 6 1005 496

4.7 0.43” 0.25 0.022

290 135f 231 121

Substrate Bz-Gly-Lys Bz-Gly-Arg Bz-Ala-Lys Bz-Ala-Arg

B

489

STUDIES

4 Determined by b Reported value c Reported value d Reported value e Reported value f Reported value

titration at pH 7.6 on a pa-stat. of 1.4 mM (23). of 0.19 mM (2) and 0.28-0.31 InM (3). of 12.0 ?nM (23). of 0.23 mM (24). of 60 s-l (24).

B

L,Kn 62 310 924 5500

490

MCKAY,

PHELAN, TABLE

INHIBITORS

Dicarboxylic acids DGGPSA DL-GEMSA DL-GPMSA DL-APMSA DL-AEMSA DL-BzlSA Monocarboxylic L-Arg L-Lys

II

OF CARBOXYPEPTIDASES

Human carboxypeptidase B” Ki (PM)

Inhibitor

AND PLUMMER

0.8 0.5 3.0 4.0 30.0 10.0

Human carboxypeptidase N* Ki (PM)

Bovine carboxypeptidase B” Ki (PM)

1.0 1.0 1.3 25.0 220.0d >10,000”

3.0 4.0” 15.0” 8.0C 75.0” 35.0”

acids 360.0 260.0

360.0 A

-f -f

a Substrate 1 mM Bz-Gly-Arg. * Substrate 1 mM Bz-Ala-Lys. c Data of McKay and Plummer (8). d Substrate Bz-Gly-Lys. e No inhibition at 10 mM. f No inhibition at 1 mM.

for GPSA or GEMSA on all three enzymes were identical. Distinct differences were evident between both pancreatic carboxypeptidases and human plasma carboxypeptidase N. All three guanidino-containing derivatives were efficient inhibitors of carboxypeptidase N. The lysine analog, APMSA, was much less efficient and BzlSA, the phenylalanine analog, was not effective at all. In contrast, human carboxypeptidase B showed less difference in binding efficiency and the difference between the best inhibitors, GEMSA or GPSA, and BzlSA was only 20-fold. These results were similar to those for bovine carboxypeptidase B (8). HN

NH2

\‘/

HN \\/

I NH I (CH2J2

NH2 I NH ywz

: JHCM)"

CH I2 CHCOOH

!H~COOH

CH2CWH

GEMSA

FIG. 1. Structure GEMSA and GPSA.

GPSA

of the competitive

inhibitors

DISCUSSION

Oshima et al. (23) first demonstrated the dramatic effect of a penultimate alanine on the hydrolysis of arginine by carboxypeptidase N. They compared Z-Ala-Arg and Bz-Gly-Arg. With this in mind, we synthesized the alanyl version of hippurylarginine and hippuryl-lysine. The data in Table I illustrate the utility of these new substrates. The use of Bz-Ala-Lys with carboxypeptidase N was especially advantageous due to the low amounts of the enzyme available. While this study was in progress, Moore and Benoiton (26) published a study comparing the efficiency of Bz-Ala-Lys and a series of Bz-Glyderivatives with porcine carboxypeptidase B. Similar results were obtained to ours on comparing the effect of the a-methyl group. The K, value of Bz-Ala-Lys was substantially lower than Bz-Gly-Lys and the kcatlK,,, was higher, indicative of a preference of all carboxypeptidases for a penultimate alanine residue. We also synthesized Bz-Val-Lys, Bz-Val-Arg, and Bz-Leu-Arg and found them to be inferior to Bz-Gly-derivatives as substrates. Apparently, increasing the size of the side

HUMAN

CARBOXYPEPTIDASES

9.3

0

B AND N: COMPARATIVE

0.5

1.0

1.5

STUDIES

491

2.0

GPSA (#I) FIG. 2. Dixon plot of the rate of hydrolysis of Bz-Ala- Arg (in 0.05 M Tris-HCl, pH 7.6,0.5 M in NaCl at 37°C) by human pancreatic carboxypeptidase B (at 1.44 x IO-” M) as a function of concentration of GPSA. Substrate concentrations are 1.0 mM (O), 0.5 mM (A), and 0.3 mM (0). Inset Cornish-Bowden plot of the hydrolysis of Bz-Ala-Lys by human plasma carboxypeptidase N as a function of concentration of GPSA at substrate concentrations of 1 mM (O), 0.5 mM (A), and 0.25 mM (0). Conditions are as in larger figure. Enzyme concentration was 1.78 x 1Om8M.

chain significantly beyond a methylene group diminishes the efficiency of the compounds as substrates. Byers and Wolfenden (27) demonstrated that BzlSA was a very potent competitive inhibitor of carboxypeptidase A. They termed the inhibitor a “bi-product analogue” that structurally resembles the products of hydrolysis of C-terminal phenylalanine peptides. We have prepared (8) a series of sulfur-containing bi-product analogs of lysine and arginine that were shown to be the most effective inhibitors known for bovine carboxypeptidase B. In addition, we have now synthesized the all-carbon analog of GEMSA (GPSA) to reaffirm the efficacy of a sulfur replacing a methylene in the P-position. The results listed in Table II demonstrate that the bi-product analogs of arginine are the most effective inhibitors to date of either human carboxypeptidase B or N. The sulfur-containing derivative (GEMSA) and an all-carbon derivative (GPSA) were equally efficient as competitive inhibitors.

The results with the series of inhibitors demonstrate similar active center binding for both bovine and human pancreatic carboxypeptidase Bs. However, the larger molecular weight human plasma carboxypeptidase N shows a strong preference for guanidino analogs. Even GPMSA, the homo-arginine version of GEMSA, is an efficient inhibitor. Moreover, the lysyl biproduct analog derivative, APMSA, was much less efficient, and AEMSA, the biproduct analog of lysine having one less carbon, was no better an inhibitor than L-lysine itself. Furthermore, while both human and bovine carboxypeptidase B, as well as porcine (28), will bind BzlSA efficiently, human carboxypeptidase N apparently binds the acid inefficiently, if at all. An earlier report (5) that GEMSA inhibited carboxypeptidase N with a Ki of approximately 8 x lo+ M was in error and is corrected in Table II. The availability of very efficient competitive inhibitors of carboxypeptidase N allows them to be tested as bradykinin potentiat-

492

MCKAY,

PHELAN,

ing agents. At present the most potent agents are bradykinin potentiating peptide (29) and its derivatives and also the mercaptoalkanoyl derivatives of proline (30). Both are specific inhibitors for angiotensin I converting enzymes and do not inhibit carboxypeptidase N. GEMSA, GPSA, and APMSA were not inhibitory to angiotensin I converting enzyme 5,6 at 1 InM concentrations. They should prove useful in determining whether carboxypeptidase N-type enzymes are important as inactivators of bradykinin in tissues as well as in plasma. Precise determination of relative contributions of different kininases to bradykinin degradation has lagged due to the lack of antagonists specific for carboxypeptidase N. ACKNOWLEDGMENTS We thank William Lawson and Thomas Ryan for advice on synthetic procedures and kinetic interpretations. We thank Mr. Matthew Kimmel for the preparation of human pancreatic carboxypeptidase B and the Northeastern New York Red Cross Blood Center in Albany, New York for pooled, outdated, human blood plasma. REFERENCES 1. WALDSCHMIDT-LEITZ, E., ZIEGLER, F., SCHAFFNER, A., AND WEIL, L. (19%). 2. t’hysicd. Chem. 197, 219-236. 2. BRODRICK, J. W., GEOKAS, M. C., AND LARGEMAN,

C. (1976) Biochim. 3.

4. 5. 6.

Biophys. Acta 452, 468-

481. MARINKOVIC, D. V., MARINKOVIC, J. N., ERDOS, E. G., AND ROBINSON, C. J. G. (1977) Biochem. J. 163, 253-260. KEMMLER, W., PETERSON, J. D., AND STEINER, D. F. (1971) J. Biol. Chem. 246, 6786-6791. PLUMMER, T. H., Jr., AND HURWITZ, M. Y. (1978) J. Biol. Chem. 253, 3907-3912. ERDOS, E. G., AND SLOANE, E. M. (1962) Biochem. Pharmacol. 11, 585-592.

5E. G. ErdBs, GEMSA and human kidney angiotensin I converting enzyme, personal communication. @A. W. Phelan, GEMSA, GPSA, APMSA, and rabbit lung angiotensin I converting enzyme, unpublished observations.

AND

7.

PLUMMER BOKISCH,

V. A., AND MOLLER-EBERHARD, H. J. Invest. 49, 2427-2436. MCKAY, T. J., AND PLUMMER, T. H., Jr. (1978) Biochemistry 17,401-405. SOKO~VSKY, RI., AND ZISAPEL, N. (19’71) Biochim. Biophys. Acta 251, 203-206. PLUMMER, T. H., JR. (1971) J. Biol. Chem. 246,

(1970) J. Clin.

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‘. 1o ’ 2930-2935. 11. PLUMMER, T.

H., JR. (1969) J. Biol. Chem. 244, 5246-5253. 12. Cox, D. J., WINTERSBERGER, E., AND NEURATH, H. (1962) Biochemistry 1, 1078-1082. 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 14. WOLFF, E. C., SCHIRMER, E. W., ANDFOLK, J. E. (1962) J. Biol. Chem. 237, 3094-3099. i5 KYCIA, J. H., ELZINGA, M., ALONZO, N., AND ’ HIRS, C. H. W. (1968) Arch. Biochem. Biophys. 123, 336-342. H., AND BURK, D. (1934) J. Amer. 16. LINEWEAVER,

Chem. Sot. 56, 658-666. I. H. (1975) Enzyme Kinetics, p. 210, 17. SEGAL, Wiley, New York. J. 55, 170-171. 18. DIXON, M. (1953) Biochem. N., NODA, K., AND ANFINSEN, C. B. lg. IZUMIYA, (1971) Arch. Biochem. Biophys. 144, 237-244. Y. S., FEIGENBAUM, A. M., DE 20. KLAUSNER, GROOT, N., AND HOCHBERG, A. A. (1978)Arch. Biochem. Biophys. 185, 151-155. G., AND VISWANATHA, T. (1974) 21. TOMLINSON, Anal. Biochem. 60, 15-24. 22 MOORE, S. (1968) J. Biol. Chem. 243,6281-6283. G., KATO, J., AND ERD~S, E. G. (1975) 23: OSHIMA, Arch. Biochem. Biophys. 170, 132-138. 24 ALTER, G. M., LEUSSING, D. L., NEURATH, H., AND VALLEE, B. L. (1977) Biochemistry 16, 3663-3668. 25 CORNISH-BOWDEN, A. (1974) Biochem. J. 137, 143- 144. 26 MOORE, G. J., AND BENOITON, N. L. (1978) ’ Canad. J. Biochem. 56, 315-318. 27 BYERS, L. D., AND WOLFENDEN, R. (1973) Biochemistry 12, 2070-2078. 28 AND SOKOLOVSKY, M. (1974) ZISAPEL, N., Biochem. Biophys. Res. Commun. 58, .951959. S. H. (1965) Brit. J. Pharmacol. 29. FERREIRA, Chemother. 24, 163-169. D. W., CHEUNG, H. S., SABO, E. F., 30. CUSHMAN, AND ONDETTI, M. A. (1977) Biochemistry 16, 5484-5491.