Compared action of neutrophil proteinase 3 and elastase on model substrates. Favorable effect of S’-P’ interactions on proteinase 3 catalysis

Neutrophil proteinase 3 (Pr3) and elastase (NE) may cause lung tissue destruction in emphysema and cystic fibrosis. These serine proteinases have similar P1 specifities. 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 theoretically bind at S5 – S’4 . Most p -nitroanilide substrates are turned over slowly by Pr3 as compared to 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.

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].

INTRODUCTION
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 ca. 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 independentlydiscovered proteins: (i) myeloblastin which regulates the growth and differentiation of leukemic cells, (ii) p29b which has microbicidal activity, (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 may thus, in concert with NE and cathepsin G, be responsible for lung tissue destruction occurring in emphysema and cystic fibrosis.
While 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. 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 nM and 60 nM, respectively. For the less sensitive substrates 10 to 50 -fold higher enzyme concentrations were used. The kinetic parameters k cat and K m were calculated from nonlinear least square fits to the Michaelis-Menten equation (Enzfitter software). The Pr3-catalyzed hydrolysis of Suc-Ala 4 -pNA was so slow that k cat and K m could not be determined separately. We therefore recorded the full hydrolysis of 10 -30 µM Suc-Ala 4 -pNA by 3.3 µM Pr3. The k cat /K m ratio for this enzymesubstrate pair was calculated from the progress curve as outlined below for the fluorogenic substrates.
The cleavage of the fluorogenic substrates was monitored at λ ex = 328 nm and λ 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 k cat and K m . The hydrolysis of the fluorogenic substrates was therefore

Site of substrate cleavage
The p-nitroanilide substrates (2µM) dissolved in the above buffer + 5% dimethylformamide were reacted with 0.3 -15 µM enzyme in a total volume of 1 ml. After about 20% substrate hydrolysis, the reaction was stopped with 10 µl TFA, the medium was diluted 200-fold with buffer A (buffer + 0.1% TFA) and 40 µl of this dilution was applied to a 3.9 x 100 mm C18 Novo-pack Column equilibrated with buffer A. A linear gradient formed with buffer A and buffer B (0.1% TFA 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 µΜ enzyme in a total volume of one ml. After about 50% hydrolysis, the reaction was stopped with 30 µl 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.

Compared action of Pr3 and NE on acyl-tetrapeptide p-nitroanilides
All 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 k cat /K m 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 due to differences in k cat . 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 Orn) is hydrolyzed by either NE or Pr3. The poor catalytic power of Pr3 is not due to non productive enzyme-substrate binding since HPLC analyses showed that the only reaction products resulting from partial hydrolysis by Pr3 (or NE) were acyl-tetrapeptides + pnitroaniline, which indicates that binding involves subsites S 5 to S ' 1 of the active center.
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 k cat 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 Cterminus, respectively. For reasons given in the experimental section, k cat and K m could not be determined separately. Their ratio is, however, meaningful since (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], (ii) it is a good measure of specificity [16].

DISCUSSION
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 later is formed of a highly conserved Asp-His-Ser triad whose hydrogen bonding transforms the serine Oγ 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 Oγ of the catalytic serine [17]. Fuginaga et al. [9] have shown that Pr3 and NE have similar substrate-binding sites which accounts for their preference for small aliphatic residues at P 1 [4]. However, the use of some model substrates and inhibitors indicated that Pr3 is a much poorer catalyst than NE [4, [10][11][12]. We thought this might be due to the fact that Pr3 (but not NE) binds these compounds in a predominantly non-productive way. We therefore used a series of acyl-tetrapeptide-p-nitroanilides which all bind productively at subsites S 5 to S' 1 .
This series included substrates with P 2 = Asp or Glu to explore the possibility of electrostatic S 2 -P 2 interactions [9]. Since the catalytic differences persisted for most of these compounds, we hypothesized that occupancy of subsites S 5 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 : elongation of the substrates strongly increased the catalytic efficiency of Pr3 whereas it had 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 due to full occupancy of the S' region of Pr3 is more pronounced for poor p-nitroanilide substrates (nb 1, 7, 11 with P 4 = Ala ) than for good ones (nb 19, 20 with P 4 = L2p). This suggests that S 4 -P 4 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 due to occupancy of subsite S' 1 since 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 concluding that Pr3 and NE cannot be considered 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, lamanin and collagen [4].

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 k cat and K m were equal or lower than 14% and 19% respectively. n.d. = not determined.