Compared Action of Neutrophil Proteinase 3 and Elastase on Model Substrates

FAVORABLE EFFECT OF S'-P' INTERACTIONS ON PROTEINASE 3 CATALYSIS*

Catherine KoehlDagger , C. Graham Knight§, and Joseph G. BiethDagger

From the Dagger  Laboratoire d'Enzymologie, INSERM U392, Université Louis Pasteur de Strasbourg, 67400 Illkirch, France, and the § Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom

Received for publication, October 2, 2002, and in revised form, January 16, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neutrophil proteinase 3 (Pr3) and elastase (NE) may cause lung tissue destruction in emphysema and cystic fibrosis. These serine proteinases have similar P1 specificities. We have compared their catalytic activity using acyl-tetrapeptide-p-nitroanilides, which occupy the S5-S'1 subsites of their substrate binding site, and intramolecularly quenched fluorogenic heptapeptides, which bind at S5-S'4. Most p-nitroanilide substrates are turned over slowly by Pr3 as compared with NE. These differences disappear with the fluorogenic heptapeptides, some of which are hydrolyzed even faster by Pr3 than by NE. Elongation of substrates strongly increases the catalytic efficiency of Pr3, whereas it has little effect on NE catalysis. These different sensitivities to S'-P' interactions show that Pr3 and NE are not interchangeable enzymes despite their similar P1 specificity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The azurophilic granules of polymorphonuclear neutrophils contain three serine proteinases: elastase (NE),1 cathepsin G, and proteinase 3 (Pr3), which participate in lysosomal bacterial digestion and neutrophil migration through the extracellular matrix at sites of inflammation. These enzymes are ~30-kDa glycoproteins, which belong to the chymotrypsin family of serine proteinases. Pr3 is the most recently discovered, the most difficult to isolate, and hence the less well studied proteinase of the three. It is identical to three independently discovered proteins: (i) myeloblastin, which regulates the growth and differentiation of leukemic cells; (ii) p29b, which has microbicidal activity; and (iii) the target antigen of antineutrophil cytoplasmic autoantibodies detected in patients with Wegner's granulomatosis (2). Pr3 cleaves extracellular matrix proteins including elastin, type IV collagen, fibronectin, laminin, and vitronectin (3, 4). It is able to produce lung emphysema in hamsters (3), and thus, in concert with NE and cathepsin G, it may be responsible for lung tissue destruction occurring in emphysema and cystic fibrosis.

Although many model substrates have been used to map the active site of NE (5-8), literature on the substrate specificity and the catalytic activity of Pr3 is poorly documented. Fuginaga et al. (9) have shown that the two enzymes have similar substrate binding sites. This explains why both proteinases cleave the oxidized insulin A and B chains at peptide bonds involving small aliphatic amino acid residues (4). The specificity of Pr3 for non-bulky residues has been confirmed with a limited number of model substrates (4, 10-12). As a rule, these substrates were turned over at a much lower rate by Pr3 than by NE, an unexplained observation. We have undertaken the present work to understand this puzzling finding.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- NE was isolated, and active site was titrated as described previously (13). Pr3 came from Athens Research Technology (Athens, GA) and was titrated with alpha 1-proteinase inhibitor (4). This enzyme preparation was electrophoretically pure. The presence of NE was tested by reacting 0.14 µM Pr3 with increasing amounts of secretory leukoprotease inhibitor (1-8 µM, final concentration), which inhibits NE but does not inhibit Pr3 (3). The buffer was the same as that used for enzyme kinetics. After 5 min at 25 °C, 2.1 mM MeOSuc-Ala2-Pro-Val-pNA, a very sensitive NE substrate (5), was added to the mixtures and the enzymic rates were measured at 410 nm and 25 °C. The lack of enzyme activity indicated that this commercial preparation of Pr3 was free of contaminating NE. Most p-nitroanilide substrates were purchased from Bachem (Bubendorf, Switzerland). Suc-Ala2-Asp-Val-pNA, Suc-Ala2-Glu-Val-pNA, Suc-Leu-Val-Glu-Ala-pNA, Suc-Ala4-pNA, MeOSuc-L2p-Tyr-Asp-Ala-pNA, and MeOSuc-L2p-Tyr-Asp-Val-pNA were synthesized by Enzyme System Products (Livermore, CA). The intramolecularly quenched fluorogenic substrates Mca-peptide-Dpa-NH2 were synthesized using the same methodology as described earlier (14). The compounds were purified by reverse-phase chromatography, and their identity was confirmed by mass spectrometry. All of the substrates were dissolved in dimethylformamide. Their concentration was checked spectrophotometrically using epsilon 315 nm = 14,600 M-1 cm-1 and epsilon 410 nm = 7,500 M-1 cm-1 for p-nitroanilides and fluorescent substrates, respectively.

Kinetics of Substrate Hydrolysis-- All of the kinetic measurements were done at 25 °C and pH 7.4 (50 mM Hepes, 150 mM NaCl). The enzymatic reactions were initiated by adding a small volume of enzyme solution to the buffered substrate solution contained in a spectrophotometer or a fluorometer cuvette. The final concentration of dimethylformamide was 5% (v/v) throughout.

The initial rate of p-nitroanilide hydrolysis was measured at 410 nm using variable concentrations of substrate and constant concentrations of enzyme. For the most sensitive substrates, the final Pr3 and NE concentrations were 140 and 60 nM, respectively. For the less sensitive substrates, 10-50-fold higher enzyme concentrations were used. The kinetic parameters kcat and Km were calculated from non-linear least square fits to the Michaelis-Menten equation (Enzfitter software). The Pr3-catalyzed hydrolysis of Suc-Ala4-pNA was so slow that kcat and Km could not be determined separately. Therefore, we recorded the full hydrolysis of 10-30 µM Suc-Ala4-pNA by 3.3 µM Pr3. The kcat/Km ratio for this enzyme-substrate pair was calculated from the progress curve as outlined below for the fluorogenic substrates.

The cleavage of the fluorogenic substrates was monitored at lambda ex = 328 nm and lambda em = 393 nm (14) using substrate concentrations below 10 µM to avoid absorptive fluorescence quenching of the hydrolysis product. The absorptive quenching also precluded the separate measurement of kcat and Km. Therefore, the hydrolysis of the fluorogenic substrates was followed to completion. At low substrate concentrations ([S]o < Km), the Michaelis-Menten equation simplifies to v = kcat/Km × [E]o × [S]o so that the release of product P with time is a first-order reaction described by d[P]/dt = kobs [S] where kobs = kcat/Km × [E]o. The pseudo first-order rate constant kobs was calculated by non-linear regression analysis of progress curves recorded using 0.5-5 µM substrate concentrations and 0.1-1 µM enzyme concentrations. The progress curves were all first-order, and kobs did not significantly change with [S]o. This indicates that [S]o was indeed lower than Km.

Site of Substrate Cleavage-- The p-nitroanilide substrates (2 µM) dissolved in the above buffer + 5% dimethylformamide were reacted with 0.3-15 µM enzyme in a total volume of 1 ml. After ~20% substrate hydrolysis, the reaction was stopped with 10 µl of trifluoroacetic acid, the medium was diluted 200-fold with buffer A (buffer + 0.1% trifluoroacetic acid), and 40 µl of this dilution was applied to a 3.9 × 100-mm C18 Novo-pack column equilibrated with buffer A. A linear gradient formed with buffers A and B (0.1% trifluoroacetic acid in acetonitrile) was used to separate the reaction products. Elution was followed at 214 nm at a flow rate of 0.8 ml·min-1.

The fluorogenic substrates (5 µM) dissolved in the buffer + 5% dimethylformamide were reacted with 30 nM to 1.6 µM enzyme in a total volume of 1 ml. After ~50% hydrolysis, the reaction was stopped with 30 ml of glacial acetic acid. The digests were absorbed on C18 reverse-phase cartridges, washed with 5% acetonitrile to remove the salts, and then the peptide fragments eluted with 60% acetonitrile. The eluant mixtures were rotary-evaporated to dryness and taken up in 0.1 ml of 60% acetonitrile. The fragments were identified by electrospray ionization mass spectrometry. The fragment signals were 10-100 times that of the background.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Compared Action of Pr3 and NE on Acyl-tetrapeptide p-Nitroanilides-- All of the substrates listed in Table I are hydrolyzed more or less efficiently by NE, but many of them are more resistant to Pr3 than to NE. The proteolytic coefficient kcat/Km may be up to 380-fold lower for Pr3 than for NE (see substrate 1). For compound 8, the best NE substrate (5), the difference is 100-fold. With a few exceptions, the differences in reactivity are because of differences in kcat. Very poor NE substrates are not hydrolyzed at all by Pr3 (see nb 10, 11, 13). Also, none of the substrates Suc-Ala-Ala-Pro-X-pNA (X = Asp, Glu, Lys, and ornithine) is hydrolyzed by either NE or Pr3. The poor catalytic power of Pr3 is not the result of non-productive enzyme-substrate binding because high pressure liquid chromatography analyses showed that the only reaction products resulting from partial hydrolysis by Pr3 (or NE) were acyl-tetrapeptides + p-nitroaniline, which indicates that binding involves subsites S5 to S'12 of the active center.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Kinetics of the hydrolysis of acyl-tetrapeptide p-nitroanilides by Pr3 and NE at pH 7.4 and 25 °C
For the sake of clarity, the kinetic constants are rounded off and the errors attached to them are not given. The errors on kcat and Km were equal or lower than 14 and 19%, respectively. ND, not determined.

A number of compounds are cleaved by Pr3 and NE with comparable proteolytic coefficients. These are either poor (nb 12, 14, 15) or good NE substrates (nb 18-20). Compound 19 is the only p-nitroanilide whose kcat for Pr3 is significantly higher than that for NE.

Compared Action of Pr3 and NE on Fluorogenic Peptides-- We have synthesized a number of heptapeptides to cover both S and S' subsites of the active center of Pr3 and NE. To follow their hydrolysis using fluorescence emission, we have included the fluorescent label Mca and the fluorescence quencher Dpa (14) at their N and C termini, respectively. For reasons given under "Experimental Procedures," kcat and Km could not be determined separately. However, their ratio is meaningful because (i) it represents the second-order acylation rate constant whether acylation is rate-limiting or not and whether substrate binding is productive or not (15), and because (ii) it is a good measure of specificity (16).

The cleavage sites of the fluorogenic substrates were determined by electrospray ionization mass spectrometry. The masses (Da) of the N-terminal peptides generated by Pr3 and NE were 609.5 and 609.3 for substrate 21 (theoretical mass of Mca-Ala-Ala-Pro-Leu = 609.7); 547.1 and 545 for substrate 22 (Mca-Ala4 = 541.5); 839.7 and 839.4 for substrate 24 (Mca-L2p-Tyr-Asp-Ala = 839.8); 867.7 and 865.5 for substrate 25 (Mca-L2p-Tyr-Asp-Val = 867.9); 880.8 and 879.5 for substrate 26 (Mca-L2p-Tyr-Asp-Ile = 881.9); and 789.3 and 788.2 for substrate 27 (Mca-L2p-Val-Glu-Ala = 789.8). The N-terminal peptide generated by the action of NE on substrate 23 had a mass of 595.6 (Mca-Ala2-Pro-Val = 596.6). The Pr3 + substrate 23 mixture did not give a clear-cut mass profile because of paucity of the material.

Table II shows that Pr3 hydrolyzes most fluorogenic substrates very rapidly. It is even more effective than NE on compounds 24-27. Table II also compares the proteolytic coefficients of Pr3 and NE on six heptapeptidic and tetrapeptidic substrates encompassing identical N-terminal tetrapeptidic sequences. It can be seen that the catalytic activity of Pr3 dramatically increases with peptide chain elongation, whereas that of NE does not significantly vary with substrate length.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Compared catalytic activity of Pr3 and NE on intramolecularly quenched fluorogenic substrates at pH 7.4 and 25 °C
The error on kcat/Km is <=  20%. The table also compares fluorogenic (Fluo) and p-nitroanilide substrates (from Table I) encompassing identical N-terminal tetrapeptidic sequences. Fluo/pNA means kcat/Km of fluorogenic substrate/kcat/Km of pNA substrates. The arrow indicates the cleavage points. n.h. = no hydrolysis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pr3 and NE belong to the chymotrypsin-like family of serine proteinases whose active site is composed of a substrate-binding site responsible for specificity and of a catalytic site responsible for substrate hydrolysis. The latter comprises a highly conserved Asp-His-Ser triad whose hydrogen bonding transforms the serine Ogamma into a powerful nucleophile that attacks the scissile peptide bond of the substrate. The efficacy of catalysis is due in part to a precise binding of substrate with resultant proper orientation of the scissile bond with respect to Ogamma of the catalytic serine (17). Fuginaga et al. (9) have shown that Pr3 and NE have similar substrate-binding sites that account for their preference for small aliphatic residues at P1 (4). However, the use of some model substrates and inhibitors indicated that Pr3 is a much poorer catalyst than NE (4, 10-12). We thought this might be because of the fact that Pr3 (but not NE) binds these compounds in a predominantly non-productive way. Therefore, we used a series of acyl-tetrapeptide-p-nitroanilides, which all bind productively at subsites S5 to S'1. This series included substrates with P2 = Asp or Glu to explore the possibility of electrostatic S2-P2 interactions (9). Because the catalytic differences persisted for most of these compounds, we hypothesized that the occupancy of subsites S5 to S'1 of Pr3 may not be sufficient to align the substrate in a position favorable for efficient catalysis, whereas it may be sufficient in the case of NE. The use of fluorogenic heptapeptides confirmed our hypothesis that elongation of the substrates strongly increased the catalytic efficiency of Pr3, whereas it had a much less effect on NE catalysis. Thus, the poor catalytic activity of Pr3 on tetrapeptidic substrates is no longer observed with heptapeptidic substrates, some of which are turned over even more rapidly by Pr3 than by NE. It is interesting to note that the rate-enhancing effect attributed to full occupancy of the S' region of Pr3 is more pronounced for poor p-nitroanilide substrates (nb 1, 7, 11 with P4 = Ala) than for good ones (nb 19, 20 with P4 = L2p). This finding suggests that S4-P4 and S'-P' interactions play a complementary role in binding substrates nb 19 and 20 in a position favorable for catalysis.

Whereas many studies have probed the S region of the substrate binding site of serine proteinases, literature is poorly documented on the S'-binding region (19). Stein and Strimpler (7) have shown that the favorable effect of S'-P' interaction on NE catalysis (18) is mainly the result of occupancy of subsite S'1 because there are no important binding interactions available past S'1. This is in agreement with our data showing that elongation of the NE substrates beyond S'1 has no significant effect on catalysis.

The present work is the first report on S'-P' interactions in Pr3. It explains why p-nitroanilides (4, 12) and thiobenzyl esters (10, 11) were found to be poor substrates of Pr3. It also allows the conclusion that Pr3 and NE cannot be considered as two interchangeable enzymes despite their common specificity for small aliphatic amino acid residues. Indeed, the divergent effects of S'-P' binding may lead to different specificities on biological protein substrates. In this context, it is noteworthy that Pr3 and NE show differences in the digestion pattern of fibronectin, laminin, and collagen (4).

    ACKNOWLEDGEMENTS

We thank Dr. Christian Boudier for assistance and the French cystic fibrosis association, Vaincre la Mucoviscidose, for financial support.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: INSERM U 392, Faculté de Pharmacie, 74 route du Rhin, 67400 Illkirch, France. Tel.: 33-3-90-24-41-82; Fax: 33-3-90-24-43-08; E-mail: jgbieth@aspirine.u-strasbg.fr.

Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M210074200

2 The S and S' subsites of the substrate binding site and the corresponding P and P' residues of the substrate are labeled according to the nomenclature of Schechter and Berger (1).

    ABBREVIATIONS

The abbreviations used are: NE, human neutrophil elastase; Pr3, human neutrophil proteinase 3; Suc, succinyl; MeOSuc, methoxysuccinyl; Ac, acetyl; Boc, tert-butyloxycarbonyl; pNA, p-nitroanilide; L2p, lysyl-(2-picolinoyl); Mca, (7-methoxycoumarin-4-yl)acetyl; Dpa, N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl; nb, number.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162[Medline] [Order article via Infotrieve]
2. Hoidal, J. R. (1998) in Handbook of Proteolytic Enzymes (Barrett, A. J. , Rawlings, N. D. , and Woessner, J. F., Jr., eds) , pp. 62-65, Academic Press, London, United Kingdom
3. Kao, R. C., Wehner, N. G., Skubitz, K. M., Gray, B. H., and Hoidal, J. R. (1988) J. Clin. Invest. 82, 1963-1973[Medline] [Order article via Infotrieve]
4. Rao, N., Wehner, N., Marshall, B., Gray, W., Gray, B., and Hoidal, J. (1991) J. Biol. Chem. 266, 9540-9548[Abstract/Free Full Text]
5. Nakajima, K., and Powers, J. C. (1979) J. Biol. Chem. 254, 4027-4032[Medline] [Order article via Infotrieve]
6. Yasutake, A., and Powers, J. C. (1981) Biochemistry 20, 3675-3679[Medline] [Order article via Infotrieve]
7. Stein, R. L., and Strimpler, A. M. (1987) Biochemistry 26, 2238-2242[Medline] [Order article via Infotrieve]
8. Lestienne, P., and Bieth, J. G. (1980) J. Biol. Chem. 255, 9289-9294[Free Full Text]
9. Fujinaga, M., Chernaia, M. M., Halenbeck, R., Koths, K., and James, M. N. G. (1996) J. Mol. Biol. 261, 267-278[CrossRef][Medline] [Order article via Infotrieve]
10. Brubaker, M. J., Groutas, W. C., Hoidal, J. R., and Rao, N. V. (1992) Biochem. Biophys. Res. Commun. 188, 1318-1324[Medline] [Order article via Infotrieve]
11. Kam, C. M., Kerrigan, J. E., Dolman, K. M., Goldschmeding, R., Von dem Borne, A. E. G. Kr., and Powers, J. C. (1992) FEBS Lett. 297, 119-123[CrossRef][Medline] [Order article via Infotrieve]
12. Früh, H., Kostoulas, G., Michel, B. A., and Baici, A. (1996) Biol. Chem. 377, 579-586[Medline] [Order article via Infotrieve]
13. Boudier, C., and Bieth, J. G. (2001) Biochemistry 40, 9962-9967[CrossRef][Medline] [Order article via Infotrieve]
14. Knight, C. G., Willenbrock, F., and Murphy, G. (1992) FEBS Lett. 296, 263-266[CrossRef][Medline] [Order article via Infotrieve]
15. Bender, M. L., and Kezdy, F. J. (1965) Annu. Rev. Biochem. 34, 49-76[Medline] [Order article via Infotrieve]
16. Fersht, A. R. (1974) Proc. R. Soc. Lond. B 187, 397-407[Medline] [Order article via Infotrieve]
17. Perona, J. J., and Craik, C. S. (1995) Protein Sci. 4, 337-360[Abstract/Free Full Text]
18. Mc Rae, B., Nakajima, K., Travis, J., and Powers, J. C. (1980) Biochemistry 19, 3973-3978[Medline] [Order article via Infotrieve]
19. Hedstrom, L. (2002) Chem. Rev. 102, 4501-4524[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.