Elastase substrate specificity tailored through substrate-assisted catalysis and phage display

William Dall'Acqua, Cornelia Halin1, Maria L. Rodrigues and Paul Carter2

Department of Molecular Oncology, Genentech, Inc., 1 DNA Way,South San Francisco, CA 94080, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The catalytic histidine of human neutrophil elastase was replaced with alanine (H57A) to determine if a substrate histidine could substitute for the missing catalytic group—`substrate-assisted catalysis'. H57A and wild-type elastase were recovered directly from Pichia pastoris following expression from a synthetic gene lacking the elastase pro sequence, thereby obviating the need for zymogen activation. Potential histidine-containing substrates for H57A elastase were identified from a phage library of randomized sequences. One such sequence, REHVVY, was cleaved by H57A elastase with a catalytic efficiency, kcat/KM, of 2800 s–1 M–1, that is within 160-fold of wild-type elastase. In contrast, wild-type but not H57A elastase cleaved the related non-histidine containing sequence, REAVVY. Ten different histidine-containing linkers were cleaved by H57A elastase. In addition to the requirement for a P2 histidine, significant preferences were observed at other subsites including valine or threonine at P1, and methionine or arginine at P4. A designed sequence, MEHVVY, containing the preferred residues identified at each subsite proved to be a more favorable substrate than any of the phage-derived sequences. Extension of substrate-assisted catalysis to elastase suggests that this engineering strategy may be widely applicable to other serine proteases thereby creating a family of highly specific histidine-dependant proteases.

Keywords: human neutrophil elastase/phage display/protease specificity/substrate-assisted catalysis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Site-specific proteolysis of peptides and proteins is a much sought-after goal in biochemistry and biotechnology. Unfortunately many proteases have broad substrate specificity rendering them seldom if ever useful for this task. More selective proteases, such as factor Xa, enterokinase, caspases and various viral proteases, cleave some proteins at unique sites but will also proteolyze at closely related sequences (Carter, 1990). The very limited repertoire of sequences that can be specifically cleaved is being slowly expanded through the discovery of new proteases as well as by modifying the substrate specificity of known proteases.

Substrate-assisted catalysis, as shown with the serine proteases subtilisin BPN' (Carter and Wells, 1987Go) and trypsin (Corey et al., 1995Go), is a strategy for engineering proteases with exquisite substrate specificity by replacing part of the catalytic machinery with a similar functional group in the substrate. The activity of subtilisin BPN' against peptidyl p-nitroanilide substrates is reduced by ~106-fold by replacing the catalytic triad histidine with alanine, H64A (Carter and Wells, 1987Go). The function of this missing catalytic group can be partially restored by a histidine at either the P2 (Carter and Wells, 1987Go) or P1' (Carter et al., 1989Go; Matthews and Wells, 1993Go) positions (Schechter and Berger, 1968Go) of the substrate. The catalytic efficiency (kcat/KM) of substrate-assisted catalysis by H64A subtilisin has been increased 150-fold by rational enzyme design and substrate optimization (Carter et al., 1989Go; Carter et al., 1991Go). Favorable substrates have also been identified using substrate phage (Matthews and Wells, 1993Go).

Human neutrophil elastase, hereafter referred to as elastase, is a serine protease that is expressed and stored in human neutrophils and released upon neutrophil activation (Dewald et al., 1975Go). Elastase shares a virtually identical three-dimensional fold with trypsin and chymotrypsin which is different from that of subtilisin BPN'. Nevertheless the catalytic triad of all of these serine proteases are virtually superimposable (Corey and Craik, 1992Go). Here we test the concept of substrate-assisted catalysis in elastase by using substrate phage (Matthews and Wells, 1993Go) to identify favorable substrates for H57A elastase (chymotrypsin numbering scheme). Phage-derived and related designed sequences were then evaluated as substrates in the context of fusion proteins. This work necessitated the development of a direct method for the recombinant production of active elastase.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Computer modeling

The three-dimensional structure of elastase complexed with turkey ovomucoid inhibitor third domain (Bode et al., 1986Go) was analyzed using Insight II 95.0 (Molecular Simulations, San Diego, CA) running on an Indigo work station (Silicon Graphics, Mountain View, CA). The P2 and P1' residues of the inhibitor were replaced by histidine. These modeled histidines were adjusted manually to mimic the interaction between the catalytic H57 and the other catalytic triad residues, S195 and D102 (chymotrypsin numbering scheme). Hydrogen bond angles and distances as well as angles between the catalytic or substrate histidines and the other catalytic groups were estimated as described (Carter and Wells, 1987Go).

Construction of synthetic elastase gene

A gene encoding elastase was assembled from 24 synthetic oligonucleotides (54–68mer) sharing 4 base pair overlaps with adjacent oligonucleotides as described (Dennis et al., 1993Go). The H57A mutation and a hexahistidine-encoding sequence were installed by site-directed mutagenesis (Kunkel et al., 1987Go) and the nucleotide sequence verified. Wild-type and H57A elastase genes were cloned as XhoI–EcoRI fragments into the P.pastoris expression vector, pPIC9 (Invitrogen, Carlsbad, CA) to create pPIC9WThne and pPIC9H57Ahne, respectively. The H57A elastase gene was also cloned into the mammalian expression vector, pRK5 (Suva et al., 1987Go), to create pRK5H57Ahne.

Elastase production

A small quantity (~50 µg) of H57A elastase suitable for panning was obtained following transient transfection of human embryonic kidney 293 cells with pRK5H57Ahne (Gorman et al., 1990Go). The conditioned media was ultrafiltrated, dialyzed into 50 mM Tris–HCl (pH 8.0), 1 M NaCl (buffer A) and applied to a 1 ml nickel nitrilotriacetic acid (Ni2+-NTA) Superflow column (Qiagen, Valencia, CA). The resin was washed with 20 ml buffer A containing 10 mM imidazole and the H57A elastase eluted with a gradient of 10–200 mM imidazole in buffer A. Purified elastase from 293 cells and P.pastoris (below) was dialyzed four times against 100 mM Tris–HCl (pH 8.0), flash frozen and stored at –70°C. The yield of elastase was estimated by amino acid hydrolysis and ELISA.

Larger quantities of H57A elastase (~500 µg) together with the wild-type enzyme (~50 µg) were obtained following expression in P.pastoris GS115 cells using commercially available protocols (Invitrogen) and used for fusion protein proteolysis studies. Elastase was affinity-purified from culture supernatants using 5 mg each of the anti-elastase monoclonal antibodies, 4E4 and 5A1 (Genentech), immobilized on 10 ml CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech). The resin was washed with 500 ml phosphate-buffered saline (pH 7.4) and the elastase eluted with 50 mM triethylamine (pH 11.0). Eluted fractions were neutralized with 1.0 M Tris–HCl (pH 7.0), adjusted to 5 mM EDTA (0.5 h, 4°C) then 50 mM MgCl2 (0.5 h, 4°C). The elastase was then adjusted to 25 mM imidazole and applied to a 3 ml Ni2+-NTA column. The resin was washed with 60ml 25mM imidazole in 50 mM Tris–HCl (pH 8.0) and the elastase eluted with 350 mM imidazole in 50 mM Tris–HCl (pH 7.5).

Elastase ELISA

Elastase purified from human neutrophils (Calbiochem, La Jolla, CA) was quantified by amino acid analysis to provide an ELISA standard. Individual wells of a 96-well Immuno plate (Nunc, Rochester, NY) were coated with 1 µg bovine pancreatic trypsin inhibitor (Sigma, St Louis, MO) or 1 µg 4E4. The plates were blocked with 1% (w/v) bovine serum albumin (Intergen, Purchase, NY), incubated with samples or standards, then with a sheep anti-elastase polyclonal antibody (Biodesign International, Kennebunk, ME) followed by a horse radish peroxidase conjugate of a mouse anti-sheep polyclonal antibody (Sigma). Peroxidase activity was detected with o-phenylenediamine dihydrochloride (Sigma) and the reaction was quenched with 4 M HCl. The absorbance at 450 nm was measured with a Spectramax 340 plate reader and SoftMaxPro 1.2.0 software (Molecular Devices, Sunnyvale, CA). The signal response was found to be linear over the range 1–10 ng/ml and 1–50 ng/ml elastase for 4E4 and BPTI-coated wells, respectively. Similar results were obtained with both BPTI and 4E4 coats, suggesting that the recovered elastase is fully active.

Construction and selection of substrate phage libraries

A phage library was constructed starting from the template phGH-LIB-G3 (Matthews and Wells, 1993Go) by site-directed mutagenesis (Kunkel et al., 1987Go) using the oligonucleotide 5'-AGCTGTGGCCCAGGTGGTNNSNNSCACNNSNNSNN-SGGTGGTCCAGGGTCGACTGGCGGTGGCTCT-3', where N = T, C, G or A and S = G or C. The template contains eight stop codons and introduces a frame shift between human growth hormone (hGH) and gIII so that only mutagenized phagemids will give rise to hGH-displaying phage. Correctly mutagenized phage contain the sequence GPGGX3HX2GGPG. The library was propagated and panned on hGH receptor as previously described (Matthews and Wells, 1993Go) with the following modifications. Phage were eluted with 1 µM H57A elastase derived from 293 cells (0.5 h, 25°C). Protease resistant phage still bound to the plates were then eluted with 50 mM glycine (pH 2.0). The selection procedure was then repeated six times. Clones from the protease-sensitive and protease-resistant pools were sequenced after four and seven rounds of panning.

Preparation and digestion of fusion proteins

Phagemid, pZAP, encodes a fusion protein (Z–AP) in which the synthetic Z domain of Staphylococcus aureus protein A is joined by a linker sequence to Escherichia coli alkaline phosphatase (Carter et al., 1989Go). Different linker sequences were installed by site-directed mutagenesis (Kunkel et al., 1987Go). Z–AP fusion proteins were expressed in E.coli and purified as described previously (Carter et al., 1989Go). Z–AP fusion proteins (6 µM) were digested (0.5–10 h, 37°C) with either H57A (1 µg/ml) or wild-type (20 ng/ml) elastase in 100 mM Tris–HCl (pH 8.0), 5 mM EDTA in the presence (H57A) or absence (wild-type) of 1 mM phenylmethylsulfonyl fluoride (PMSF). The digests were terminated with Tris–glycine–SDS sample buffer and analyzed on 8% SDS–polyacrylamide gels (Novex, San Diego, CA). Gels were stained for 3 h with Serva blue G (Serva, Heidelberg, Germany) and destained for 4–5 h in 10% (v/v) acetic acid 20% (v/v) ethanol. AP released was quantified by scanning laser densitometry (model GS-670 with Molecular Analyst 1.1 software, Bio-Rad). Initial rates of Z–AP cleavage were estimated from five to eight successive time points, under conditions where <=10% of the fusion protein was digested. For some fusion proteins, initial cleavage rates were estimated over a range of substrate concentrations (0.02–4 µM) and kcat and KM values estimated by a non-linear least squares fit of the data to the Michaelis–Menten equation using Kaleidagraph 3.0.8 (Synergy Software, Reading, PA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Structural mimicry of elastase catalytic histidine by substrate histidines

P2 and P1' substrate histidine residues were modeled using the structure of elastase complexed with turkey ovomucoid inhibitor (Bode et al., 1986Go). A priori the non-ionized imidazoyl ring of a substrate histidine might exist in either N{delta}1H or N{varepsilon}2H tautomers, in which N{delta}1 and N{varepsilon}2 atoms are protonated, respectively. The catalytic histidine of elastase, H57, adopts the N{delta}1H tautomer with N{varepsilon}2 poised to accept a proton from the catalytic serine, S195 (Bode et al., 1986Go). The histidine in model peptides also commonly adopt the N{delta}1H tautomer although this depends upon their local environment (Creighton, 1984). We therefore investigated the structural mimicry of the catalytic histidine by P2 and P1' substrate histidines in the more likely N{delta}1H tautomer before considering the N{varepsilon}2H tautomer.

The substrate histidines were initially modeled such that their N{delta}1 and N{varepsilon}2 atoms best approximate the position of the corresponding nitrogens from the catalytic histidine (Figure 1Go). Molecular details were investigated by comparing substrate and catalytic histidines in their torsion angles and possible hydrogen-bond interactions with other catalytic residues (Table IGo). The modeled P2 histidine has a very similar hydrogen bond angle and distance with S195 as does the catalytic histidine (model 1), albeit at the expense of a {chi}2 torsion angle that falls outside the range of observed histidine rotamers (Ponder and Richards, 1987Go). A histidine at P1' is less favorable than one at P2 in mimicking the interaction between H57 and other members of the catalytic triad (Table IGo). In particular, a P1' histidine, unlike a P2 histidine, is too distant from D102 to form a direct hydrogen bond (models 3 and 1, respectively). When the dihedral angles of the P2 (model 2) and P1' (model 4) histidines are constrained to ideality, the only plausible modeled hydrogen bond is between the P2 histidine and S195.



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Fig. 1. Molecular model showing the predicted interactions between elastase and a substrate containing histidines at P2 and P1' (red, models 1 and 3 in Table IGo, respectively) positions. The substrate (yellow) is shown together with the main chain of elastase (violet) and the catalytic triad: S195, H57 and D102 (green). Molecular modeling was based upon the X-ray crystallographic structure of elastase complexed with the turkey ovomucoid inhibitor (Bode et al., 1986Go).

 

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Table I. Bond angles and distances modeled for substrate-assisted catalysis by P2 and P1' substrate histidines in elastasea
 
Analogous models were constructed in which the substrate histidine was in the N{varepsilon}2H tautomer with the unprotonated N{delta}1H poised to accept a proton from S195. Modeling results with the N{varepsilon}2H tautomer (Table IGo, models 1–4) were broadly similar to those with the N{delta}1H tautomer (Table IGo, models 5–8). This significant structural mimicry observed between substrate and catalytic histidines encouraged us to replace the elastase catalytic histidine with alanine (H57A) and investigate the ability of P2 and P1' substrate histidines to substitute functionally for the missing catalytic group.

Elastase production

A synthetic gene was constructed for the expression of wild-type and H57A elastase (GenBank accession number AF117205, Figure 2Go). The 5' end of the elastase gene was fused to the Mat{alpha} gene to direct secretion from P.pastoris. The two residue pro sequence of elastase, S1E2, was purposely omitted in anticipation that cleavage by Kex 2 protease in vivo would generate the N-terminus found in the mature active enzyme rather than the inactive zymogen. The titers of H57A and wild-type elastase in P.pastoris reached plateaux of 1.0 µg/ml and 0.1 µg/ml, respectively, following 72 h induction with methanol. The elastase variants were affinity-purified using two anti-elastase antibodies followed by immobilized metal affinity chromatography (IMAC). H57A and wild-type elastase were recovered in up to 5% yield and >=90% homogeneity as evidenced by ELISA and SDS–PAGE, respectively (data not shown). The N-termini of H57A and wild-type elastase were found to be IVGGRRAR, consistent with Kex 2 cleavage immediately following the {alpha}-factor.



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Fig. 2. (A) Schematic representation and (B) nucleotide sequence of a synthetic gene for the expression of wild-type elastase in P.pastoris. The highlighted catalytic triad residues, H43, D90 and S175 correspond to H57, D102 and S195, respectively, in the commonly used chymotrypsin numbering scheme that is used throughout the text. Also highlighted is the N-terminal residue, I3. The elastase gene is preceded and followed by sequences encoding the Mat{alpha} peptide and hexahistidine, respectively. The 5' and 3' ends of synthetic DNA fragments used in assembling the synthetic gene are indicated in lowercase, as are amino acid residues in the Mat{alpha} peptide.

 
Identification of substrates for H57A elastase from phage library

A substrate phage library was constructed which contained variant linkers juxtaposed between hGH and the C-terminal domain of M13 gene III protein. The linker contained a histidine residue flanked by randomized residues and glycines: GGX1X2HX3X4X5GG, where X is any amino acid. This library of 6x107 clones is large enough to represent a significant proportion of the 3.4x107 (325) possible codon permutations from the NNS randomization strategy. However, sampling of the library was limited by the number of phage that were captured on immobilized hGH receptor: >=107. Phage released with H57A elastase were propagated and subjected to further rounds of panning, as were protease resistant phage. After seven but not four rounds of panning, a strong consensus sequence had emerged for the H57A sensitive (Figure 3A and CGo) but not H57A resistant clones (Figure 3B and DGo). These H57A elastase sensitive clones have predominantly R or M at position X1, E or Q at X2 and V or T at X3. The specificity is broader at the other positions with M, T, V and I being frequently found at X4 and Y, W, L and F at X5 (Figure 3CGo).



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Fig. 3. Linker sequences of substrate phage clones identified after seven rounds of panning of the X3HX2 hGH substrate library using hGH receptor. Shown are sequences of individual clones that are (A) sensitive or (B) resistant to cleavage by H57A elastase. Numbers in parentheses correspond to the occurrence frequency for clones that were found a multiple of times. Also shown are summaries of (C) H57A sensitive and (D) H57A resistant clones. Sequences are aligned relative to the fixed histidine (see Results). The highlighted residues in (D) match the abundantly found residues at corresponding positions in (C).

 
Histidine-dependant proteolysis by H57A elastase

The ability of H57A elastase to cleave sequences identified from substrate phage was investigated using Z–AP fusion proteins constructed with nine such sequences as linkers (Table IIGo, L1–L9). Nine additional fusion proteins were designed: five by permutating common residues in phage-derived sequences (L10–L14), three with a P1' histidine (L15–L17) and a control sequence lacking a histidine (L18). These 18 different Z–AP fusion proteins allowed assessment of proteolysis by H57A elastase, including histidine dependence and the subsite position of the histidine (P2 or P1') as well as specificity at other subsites (Table IIGo).


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Table II. Proteolysis of Z–AP fusion proteins by H57A and wild-type elastasea
 
Z–AP fusion proteins were secreted from E.coli grown in shake flasks, and recovered in yields of 0.3–0.8 mg/l by IgG affinity chromatography. The initial rate of cleavage of Z–AP fusion proteins (54 kDa) by H57A and wild-type elastase was determined from the release of AP (47 kDa) as followed by SDS–PAGE and scanning laser densitometry (Table IIGo). Z domain release was not followed because of its small size (7 kDa) and weak staining with Coomassie blue (Carter et al., 1989Go).

Several histidine-containing Z–AP fusion proteins were cleaved by H57A elastase including 6/9 phage-derived and 4/8 designed sequences. In each case the histidine residue was located at the P2 position as evidenced by N-terminal sequence analysis of the AP product (Table IIGo). Cleavage by H57A elastase with a P1' histidine was not detected, even for three purposely designed linkers (L15–L17) that were cleaved by wild-type elastase (Table IIGo). The absence of cleavage by H57A elastase at other histidines in Z–AP fusion proteins likely reflects that only 2/12 of these sites have favorable P1 residues (LVAH163VTS and SQEH413TGS) and these sites are at least partially buried within AP (Sowadski et al., 1985Go).

A Z–AP fusion protein containing the linker, REHVVY (L1), was readily cleaved to completion following extensive digestion with H57A or wild-type elastase (Figure 4Go). Replacement of the histidine in the linker with alanine, REAVVY (L18), abolished detectable cleavage by H57A but not wild-type elastase (Figure 4Go). Thus a P2 histidine is apparently a necessary but not sufficient condition for proteolysis by H57A but not wild-type elastase. H57A but not wild-type elastase is resistant to PMSF inhibition (Figure 4Go). Thus, the catalytic histidine is apparently required for stable sulfonylation of the active site serine as previously observed for H64A subtilisin BPN' (Carter and Wells, 1987Go).



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Fig. 4. Cleavage of Z–AP fusion proteins by H57A and wild-type elastase. Z–AP fusion proteins (4 µM) with histidine-containing (L1) or non-histidine containing (L14) linkers were digested with 50 nM H57A or 0.1 nM wild-type elastase in the absence (–) or presence (+) of 1 mM PMSF (24 h, 37°C). The bands with apparent molecular weights of 54, 47, 31 and 28 kDa represent Z–AP fusion protein, AP, H57A elastase and a minor contaminant in the fusion protein, respectively.

 
Subsite specificity of H57A and wild-type elastase

Efficient cleavage of Z–AP fusion proteins by H57A elastase apparently requires a P2 histidine as well as favorable residues at other subsites (P4 to P2') (Table IIGo). For example, valine is strongly preferred over threonine at P1 (L10 versus L2, L1 versus L8, L12 versus L14), glutamate is favored over glutamine at P3 (L1 versus L12, L5 versus L6) and methionine is preferable to arginine at P4 position (L10 versus L1, L2 versus L8). Valine is favored over isoleucine (L1 versus L5, L12 versus L6) and threonine (L1 versus L13) at the P1' position, whereas tyrosine (L1, L5), tryptophan (L11) and phenylalanine (L3) but not isoleucine (L7) are favored residues at P2'. A designed linker, MEHVVY (L10) containing the preferred residues identified at each subsite position, proved to be a more favorable substrate than any of the phage-derived sequences (L1–L9) evaluated. Beyond the P2 position, similar subsite specificity trends were observed for wild-type and H57A elastase. One exception is that threonine is slightly preferred over valine at the P1' position for wild-type elastase, whereas valine is very strongly favored over threonine at this position by H57A elastase (L13 versus L1).

The subsite specificity of H57A elastase is strongly dependent upon residues at neighboring subsites. For example, with arginine at P4, two linkers that were uncleavable with H57A elastase were converted to good substrates by replacement of P1 threonine with valine (L8 versus L1 and L14 versus L12, respectively). In contrast, in the context of the more favorable P4 residue, methionine, replacement of P1 threonine with valine resulted in a much more modest fivefold enhancement in cleavage rate (L2 versus L10). Thus installing a favorable residue at one subsite diminishes the enhancement in substrate cleavage upon subsequent installation of a highly favorable residue at a second subsite.

Comparison of proteolysis rates by H57A and wild-type elastase

The most favorable Z–AP fusion protein substrates for H57A elastase (L1, L2 and L10) are cleaved at initial rates within 100–380-fold of those observed with wild-type elastase (Table IIGo). A more detailed kinetic analysis was undertaken to distinguish between effects upon kcat and KM. Kinetic parameters for hydrolysis of Z–AP fusion proteins with histidine, REHVVY (L1), and non-histidine, REAVVY (L18), containing linkers were determined from initial cleavage rates determined over a range of substrate concentrations (Table IIIGo). The specificity constant, kcat/KM, for H57A elastase was only 160-fold lower than for the wild-type enzyme against the histidine-containing substrate (L1). This reflects an 80-fold decrease in kcat and a twofold increase in KM. In contrast, replacing the histidine in the substrate by alanine increased the activity with wild-type elastase by sevenfold and abolished detectable cleavage by H57A elastase.


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Table III. Kinetic analysis of Z–AP cleavage by H57A and wild-type elastasea
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Here we have demonstrated that the function of the catalytic histidine of human neutrophil elastase can be partially restored by a P2 substrate histidine in a mutant enzyme lacking this catalytic group (H57A) as predicted by molecular modeling (Table IGo) and originally proposed by Corey et al. (1995).

The activity of recombinant H57A and wild-type elastase was investigated using Z–AP fusion proteins rather than the more convenient and widely used peptidyl p-nitroanilide substrates. This strategy allowed direct evaluation of phage-derived sequences as potential substrates for H57A elastase. This is important since specificity determinants on the C-terminal side of the scissile bond can enhance the cleavage of peptide substrates by serine proteases (Bauer et al., 1981Go; Bizzozero and Dutler, 1987Go). Furthermore, hydrolysis of labile p-nitroanilide substrates by serine protease catalytic triad mutants is not always predictive of cleavage of peptide bonds (Corey and Craik, 1992Go). For example, H64A subtilisin variants will slowly hydrolyze non-histidine-containing peptidyl p-nitroanilide substrates whereas peptide bond cleavage is entirely histidine-dependent (Carter and Wells, 1987Go; Carter et al., 1989Go, 1991Go).

H57A elastase was found to cleave Z–AP fusion proteins with a histidine at the P2 but not P1' substrate positions (Table IIGo, Figure 4Go). A priori, this might reflect that a P2 substrate histidine mimics the catalytic histidine in facilitating both acylation and subsequently deacylation of the catalytic serine, whereas a P1' histidine is only available to participate in the acylation reaction. This explanation is unsatisfactory since subtilisin BPN' (Carter and Wells, 1987Go; Carter et al., 1989Go; Matthews and Wells, 1993Go) and trypsin (Corey et al., 1995Go) are both capable of forming an efficient peptide bond with either a P2 or a P1' histidine. More likely is that for elastase (Table IGo), in contrast to subtilisin BPN' (Matthews and Wells, 1993Go) and trypsin (Corey et al., 1995Go), a P1' histidine is much less effective than a P2 histidine in mimicking the interactions of the catalytic histidine with other catalytic triad residues (Figure 1Go). The failure to identify P1' histidine substrates might alternatively reflect other factors including library design, incomplete library sampling and inefficient selection of P1' substrates in competition with rapidly cleaved P2 histidine substrates.

Surprisingly, some phage-derived sequences (3/9) were not detectably cleaved when installed as linkers in Z–AP fusion proteins (Table IIGo, L7–L9). It seems unlikely that these substrates are not cleaved by H57A elastase but nevertheless survived seven rounds of stringent selection. A more likely possibility is that these sequences are poor substrates that are nevertheless selected during substrate phage selection since only a single turnover event is required to release phage. Consistent with this notion, these sequences (L7–L9) are very closely related to sequences that were robustly cleaved (L1–L6, L10–L13).

The design of linkers by permutating residues commonly found in phage-derived sequences (Figure 3CGo) was a successful and complimentary strategy to the use of substrate phage alone. Indeed, the most rapidly cleaved sequence (Table IIGo, L10) was obtained by design rather than directly from phage. The fact that four favorable substrates (L10–L13) were not identified directly from the substrate phage may simply reflect the number (58) of clones sequenced. Alternative explanations include deficiencies in the library or its sampling.

Several lines of evidence suggest that the activities attributed to H57A and wild-type elastase are not due to contaminating proteases. (i) H57A and wild-type elastase were purified by two independent affinity purification steps, anti-elastase then IMAC, which are highly specific for the recombinant enzymes. (ii) H57A elastase, like H64A subtilisin (Carter and Wells, 1987Go), is refractory to PMSF inhibition. In contrast, other serine proteases, including wild-type elastase, are inhibited by PMSF. This permitted PMSF addition to all assays involving H57A elastase. In addition, EDTA was added to all digestion experiments to inactivate any contaminating metallo-proteases. (iii) No cleavage of any Z–AP fusion protein was observed upon prolonged incubation in the absence of elastase. Thus the cleavage observed was not due to contaminating proteases in the Z–AP fusion protein preparations. (iv) The exquisite substrate preference of H57A elastase for a P2 histidine is unlike any known P.pastoris or E.coli protease. (v) H57A and wild-type elastase share very similar subsite specificity apart from the requirement for a P2 histidine for H57A elastase. Furthermore, the P1 subsite preference for H57A elastase is consistent with the known specificity of wild-type elastase (Powers et al., 1977Go; Stein, 1985Go; Lentini et al., 1987Go; Stein and Strimpler, 1987Go; Lu et al., 1997Go). (vi) The specific activity of the recombinant wild-type elastase agrees closely with that from elastase purified from human neutrophils (data not shown). (vii) Functional H57A and wild-type elastase were secreted directly from P.pastoris thereby obviating the need for elastase zymogen activation by the addition of exogenous protease (Okano et al., 1993Go). Thus our elastase production method avoids a source of protease contamination which might potentially confound activity measurements of catalytic triad mutants.

Our combined strategy of substrate phage augmented by rational substrate design proved to be a very effective strategy for identifying histidine-containing substrates for H57A elastase. Indeed, the activity (kcat/KM) for H57A elastase with one such substrate (L1, Table IIIGo) was only 160-fold lower than for the wild-type enzyme. Enhancements in catalytic efficiency for H57A elastase are anticipated from further substrate optimization in concert with enzyme engineering as evidenced by studies with subtilisin BPN' (Carter and Wells, 1987Go; Carter et al., 1989Go, 1991Go; Matthews and Wells, 1993Go). Thus substrate-assisted catalysis has now been demonstrated for the serine proteases elastase, subtilisin BPN' (Carter and Wells, 1987Go) and trypsin (Corey et al., 1995Go). This supports the idea that substrate-assisted catalysis may be broadly applicable to serine proteases (Corey et al., 1995Go), thereby creating a family of exquisitely specific proteases. Such engineered proteases are anticipated to be valuable additions to the protein chemist's toolbox and have potential utility in activation of peptidyl prodrugs for cancer therapy (Chakravarty et al., 1983Go).


    Acknowledgments
 
We thank Drs David Matthews and Jim Wells for kindly supplying the phagemid clone, phGH-LIB-G3, and for helpful discussions.


    Notes
 
1 Present address: ETH Hönggerberg, Zürich, CH-8093, Switzerland Back

2 To whom correspondence should be addressed; email: pjc{at}gene.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bauer,C.A., Brayer,G.D., Sielecki,A.R. and James,M.N. (1981) Eur. J. Biochem., 120, 289–294.[Abstract]

Bizzozero,S.A. and Dutler,H. (1987) Arch. Biochem. Biophys., 256, 662–676.[ISI][Medline]

Bode,W., Wei,A.Z., Huber,E.M., Meyer,E., Travis,J. and Neumann,S. (1986) EMBO J., 5, 2453–2458.[Abstract]

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Received March 29, 1999; revised May 28, 1999; accepted June 30, 1999.