Reaction Kinetics of Protease with Substrate Phage

KINETIC MODEL DEVELOPED USING STROMELYSIN*

Nikolai A. SharkovDagger, Robyn M. Davis§, John F. Reidhaar-Olson, Marc Navre, and Danying Cai||

From the Affymax Research Institute, Santa Clara, California 95051

Received for publication, December 28, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Since the first reports of the display of randomized peptide libraries on the surface of filamentous bacteriophages (1-3), phage display has become a widely adopted approach for the mapping of antibody epitopes and macromolecule-binding peptides (4-6), the selection of peptide agonists or antagonists against potential drug targets (7, 8), and the selection and optimization of sequence determinants that facilitate protein folding and stability (9-12). An important application of phage display of peptide libraries is in the selection and optimization of substrate for proteases and protein kinases (13-15). Traditionally, the sequence of a peptide substrate for protease is deduced from the natural cleavage sites of the in vivo polypeptide substrates if known. There is usually conservation of amino acid residues at the site of cleavage and in the flanking sequences. However, it has been shown that the cleavage sequences from the natural substrates are not always optimal for the enzymes when the enzymatic activities are studied in vitro with synthetic peptides (16-18). Peptide libraries generated through phage display have been instrumental in optimizing substrates and mapping the sequence preferences at both P and P' sites (13, 16-19).

The insertion of randomized sequences into protein pIII (product of gene III) of filamentous phage is the most common approach for making phage display libraries. There are up to five copies of pIII present on the surface of the phage when gene III is expressed (20). Two strategies, monovalent and multivalent display, have been reported for protease substrate phage selections (13, 19). In either case, gene III is modified such that a randomized sequence is linked to the N terminus of pIII and an affinity tag, which may be a protein or peptide epitope or a histidine tag sequence, is linked N-terminal to the random sequence (13, 19, 21, 22). The phage particle loses the epitope/tag if a peptide in the library serves as a substrate for and is cleaved by the protease of interest. Unreacted phage particles have the intact epitope/tag and can be trapped preferentially by affinity binding. The phage display approach has been successfully applied to a number of different endopeptidases (13, 16-19, 23, 24). The procedures involve iterative rounds of selection and enrichment aiming at enriching reactive phage particles containing sequences cleavable by the protease. The sequences found in the reactive clones are then transferred from the phage context and placed within a synthetic peptide or a chimeric protein for further characterization.

The assumption has been that the peptides, which were selected from the substrate phage display, would have better kcat/Km values over those not selected (13). However under normal selection conditions, the enzymes are present at nanomolar concentrations or higher whereas the phage particles at <= 1012 particles/ml, which is equivalent to 1.66 pM, or 8.3 pM if taken into account that up to five copies of pIII may be expressed on the surface of a given phage particle (20). In any case, the enzyme is in excess over the substrate, indicating a single-turnover reaction to which it is invalid to apply the equations for steady-state kinetics.

Phage is an unusual substrate for enzymes in that the phage particles present multiple copies of the substrate (the recombinant pIII) at one tip of a long cylinder (20), creating microscopic heterogeneity. It is therefore not clear whether the conventional single-turnover kinetic considerations would still apply to the reaction of substrate phage. However, a good understanding of the reaction kinetics is fundamentally important and necessary for rational experimental designs. The present study sets out to define the kinetic properties of the reaction between protease and substrate phage. Using the metalloprotease stromelysin as a model, we examine in detail the kinetics of the reaction between the enzyme and the isolated substrate phage clones. We put forth a kinetic model and further validate it using a competitive inhibitor of the enzyme. Our kinetic model will provide more rational considerations to future phage display experimental designs, which up to now have been highly empirical.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Buffers-- Streptavidin and bovine serum albumin (BSA)1 were purchased from Sigma and ECL blotting detection reagent and HRP/anti-M13 monoclonal conjugate from Amersham Pharmacia Biotech (Piscataway, NJ). The biotinylated mouse monoclonal antibody 179 (mAB179) was prepared in house. The phage clones were maintained and amplified in Escherichia coli K91 cells (19). Recombinant stromelysin was prepared as reported previously (19), and its concentration was calculated based on the total protein concentration of the protein preparation. ELISA wash solution was 0.05% Tween 20 in PBS and phage assay stop solution 0.1% BSA and 5 mM EDTA in PBS.

Phage Preparation and Phage ELISA Assay Procedures-- Typically the E. coli cells harboring the phage were cultured in 1-liter quantities, overnight at 37 °C. The phage particles were precipitated from the supernatant following a general procedure described previously (19). Phage preparations from 1-liter cultures were typically reduced to a volume of 1 ml in 25 mM Tris, pH 7.4, 20% glycerol, and 75 mM NaCl and then titered.

Plates used for ELISA assays were Microlite 2, white 96-well flatbottom plates from Dynex Technologies (Chantilly, VA). They were first coated with Streptavidin at 10 µg/ml in 10 mM PBS, pH 7.4 using 200 µl/well and incubated for 1 h at 37 °C. The next coating was done with biotinylated mAB179 at 2 µg/ml, 200 µl/well in PBS for 1 h at 37 °C. Finally, a solution of 1% BSA and 10% sucrose in PBS was added at 250 µl/well, and the plates incubated for 1 h at 37 °C. The plates were washed five times with ELISA wash solution between each coating. After the last coating, plates were aspirated and allowed to air dry.

For each ELISA assay, 200 µl of phage-containing samples were added per well, and the plate was put on a titer plate shaker at room temperature for 1 h, after which the plates were washed five times with the wash solution. HRP/anti-M13 monoclonal conjugate, diluted 40,000 times in 0.1% BSA in PBS at 200 µl/well, was added, and the plates were put on the plate shaker for 30 min at room temperature. The dilution of the HRP/Anti-M13 was optimized for each lot. Again, the plates were washed five times in wash solution. Finally, ECL blotting detection reagent was added at 200 µl/well (no incubation) and the chemiluminescence was read in Packard Topcount (Packard Instrument, Meriden, CT) in single photon count mode at 0.02 min/well.

The plate coating and the ELISA procedures were also automated. Upon completion of the transfer of the digest reaction to the ELISA plates by the Tecan (see below), a Sagian robotic arm transferred the plates to a Scitec Heidolph Titramax 100 plate shaker for the time indicated above. The Sagian robotic arm would then transfer the plates to an SLT 96 plate washer. All other reagents used for the ELISA were dispensed by Cavro syringe pumps attached to eight-pronged manifolds. The robotic arm then transferred the plates to a dedicated Packard Topcount adjacent to the robotic table. All robotics were controlled by a Pentium PC.

Quantitative Phage ELISA Assay-- The standard curves of each preparation were determined by serial diluting the phage in phage assay stop solution and running the ELISA without protease. The starting concentration for phage in the protease digest was determined from the standard curve by choosing the concentration coinciding with the uppermost part of the "useful range" (see Fig. 2). 200 µl of the stopped digest reaction (see below) were used in the ELISA assay. As a zero time, we included a tube of only phage and no enzyme. Every ELISA plate included a standard curve of phage and zero time points for quantification of every time point.

Procedures and Kinetics of Proteolytic Digest of Substrate Phage-- Phage digests for stromelysin were typically done in 150-µl reaction volumes of 20 mM HEPES, pH 7, 5 mM CaCl2, 0.02% Brij 35 containing stromelysin at concentrations between 5 nM and 1.2 µM. The reaction tubes containing the lower concentrations of stromelysin were put on a slower time course, and the tubes containing higher concentrations were put on a faster time course to sample enough points for the steepest part of the decay curve (see Fig. 3). The digest reaction was initiated by adding substrate phage at a concentration ten times higher than that determined above so that upon stopping the digest by dilution, the signal intensity would lie on the uppermost part of the useful portion of the standard curve (see Fig. 2). 15-µl time points were removed from the digest reaction and diluted 20 times in stopping solution to inactivate stromelysin. 200 µl of this solution were then removed and subjected to the quantitative phage ELISA assay to determine the concentration of the remaining substrate phage. For each stopped reaction, the ELISA assay was performed in triplicate and the averaged results graphed. The enzyme concentration-dependent phage digest experiments were performed over a course of several days, and data collected on different days were reported on the same graph.

When stromelysin inhibitor studies were performed, an enzyme concentration was chosen that would produce a well-defined digest curve reaching 100% digested within 20 min. Various concentrations of inhibitor were added to the reaction tubes and allowed to incubate with stromelysin for ~5 min before initiating the digest with a concentrated stock of phage. All linear and nonlinear regression analyses were performed using KaleidaGraph 3.09 by Synergy Software (Reading, PA).

Eventually, all of the above steps were programmed and performed with a robotic system from Scitec Inc. (Ashland, MA). The 1.2-ml digest tubes were held in a heating block that was kept at 25 °C by a circulating water bath. A Tecan liquid handler removed time points from the digest reactions and transferred them to a deep-well plate containing the stop solution. Sample was mixed three times and then waited until the next time point. When all the time points were completed, the Tecan transferred the stopped digest reaction from the deep-well plates into the ELISA plates. At this point, the ELISA assays were run either manually or robotically.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Quantitative Determination of Substrate Phage-- Fig. 1 shows the substrate phage clones used in the present study. The presence of the mAB179 epitope allowed the phage particles to be captured in wells of a microtiter plate that had been precoated with streptavidin followed by biotinylated mAB179. The captured phage was then detected using HRP-conjugated anti-M13 antibody and a chemiluminescent peroxidase substrate. When the activity of HRP was developed, the chemiluminescence signals showed a sigmoidal function with respect to phage titer on a logarithmic scale (Fig. 2A). The signals were highly specific to the phage clones containing the mAB179 epitope. The parental phage clone (fdTET) containing the wild-type pIII at 1010 cfu/ml did not cross-react at all (Fig. 2A). The change of the chemiluminescence signal was most sensitive to the change in the phage concentration on the steepest portion of the curve. This part of the curve thus served as a standard curve against which a sample of unknown phage titer could be quantified (Fig. 2B).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1.   Recombinant pIII sequences of substrate phage clones for stromelysin. The clones, created earlier (19), are identical except in the variable region where the proteolytic cleavage occurs. The arrow indicates the start of the native pIII protein, and the underlined sequence is the epitope of mAB179.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Quantitative ELISA assay curves. A, the chemiluminescence readouts in CPS are plotted as a function of the phage titer on a logarithmic scale for clone 10519 (open circle ) and the control fdTET (), and the line is the sigmoidal fit to the data using nonlinear regression. B, the useful portion of the curve serves as a standard curve for quantifying the amount of intact clone 10519 remaining in reaction mixtures.

All phage clones investigated in the present study were identical except in the protease substrate sequence (Fig. 1). We found that each clone showed noticeable but only slight variations in the shape of the standard curve when a complete titration curve was constructed for each of the clones (results not shown). To maintain the accuracy, a standard curve was constructed with the same clone that was being tested, and the curve was repeated on each of the test plates to avoid plate-to-plate variation.

Reaction Kinetics of Protease with Substrate Phage-- The time course for the proteolysis of clone 10629 by stromelysin is shown in Fig. 3. 3 × 1011 cfu/ml of 10629 was incubated with stromelysin at 25 °C. A sample was removed at each time point, and the reaction was terminated by the addition of EDTA to 5 mM final concentration. The amount of substrate phage particles remaining in the sample was determined by the quantitative phage ELISA assay as described under "Experimental Procedures." The time course was best fitted to a single-exponential equation using nonlinear regression (Equation 1),



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Time course of proteolysis of clone 10629 by stromelysin. The starting substrate phage titer was 3 × 1011 cfu/ml, and the enzyme concentrations were 40 (open circle ), 80 (), and 120 nM (). The lines are the fit to Eq. 1.


<FR><NU>[<UP>S</UP>]</NU><DE>[<UP>S</UP>]<SUB><UP>0</UP></SUB></DE></FR>×100=A<UP>e</UP><SUP>−k<SUB><UP>obs</UP></SUB><UP>*t</UP></SUP>+<UP>C</UP> (Eq. 1)
and the rate of substrate phage disappearance (kobs) was calculated. [S]0 is the starting substrate concentration. Because the phage ELISA assay determines the total substrate concentration (both enzyme-bound and unbound, see Reactions 1 and 2), the rate for substrate phage disappearance is also the measurement of the rate for product formation. That the product formation from substrate phage follows the single exponential kinetics does not seem to be limited to stromelysin, which is a metalloprotease. We have made similar observations with serine proteases including viral and bacterial enzymes.2

Pseudo First-Order Kinetic Model of Reaction between Enzyme and Substrate Phage-- Proteolysis reactions of substrate phage are normally performed with solutions containing <= 1012 cfu/ml phage (13, 19), which corresponds to an effective concentration of the recombinant pIII up to 1.66 or 8.3 pM in monovalent or multivalent display, respectively. On the other hand, the concentrations of protease used in phage display experiments are generally in nanomolar to micromolar range, which is much greater than a substrate concentration of 8.3 pM. Therefore, the reaction of the enzyme with substrate phage can be regarded as a pseudo first-order process with respect to the substrate (see Reactions 1 and 2). The general reaction scheme for protease-catalyzed substrate hydrolysis is shown in Reaction 1,


<AR><R><C><UP>E</UP>+<UP>S</UP></C><C> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>−1</SUB></LL><UL>k<SUB>1</SUB></UL></LIM></C><C><UP>E</UP> · <UP>S</UP></C><C> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>−2</SUB></LL><UL>k<SUB>2</SUB></UL></LIM></C><C><UP>E</UP> · <UP>P</UP> · <UP>P′</UP></C></R><R><C></C><C><UP>binding</UP></C><C></C><C><UP>hydrolysis</UP></C></R></AR>

<UP><SC>Reaction</SC> 1</UP>
where P and P' are hydrolyzed products of S, and P may be covalently linked to the enzyme in the case of serine or cysteine proteases. When [E]0 >> [S]0, the second-order reaction can be reduced to a pseudo first-order process as is shown in Reaction 2. 
<UP>S</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>−1</SUB></LL><UL>k<SUB>1</SUB>[<UP>E</UP>]<SUB>0</SUB></UL></LIM> <UP>E</UP> · <UP>S</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>−2</SUB></LL><UL>k<SUB>2</SUB></UL></LIM> <UP>E</UP> · <UP>P</UP> · <UP>P′</UP>

<UP><SC>Reaction</SC> 2</UP>
Our experimental data showed clearly that the product formation, reflected by substrate disappearance, was a single-exponential process (Fig. 3), which further suggests that either the substrate binding or the hydrolysis step is fast. For the pseudo first-order scheme shown in Reaction 2, the apparent single-exponential rate constant for product formation is shown in Equation 2,
k<SUB><UP>obs</UP></SUB>=k<SUB>1</SUB>[<UP>E</UP>]<SUB>0</SUB>+k<SUB>−1</SUB> (Eq. 2)
if the substrate hydrolysis is fast relative to the binding step; that is, there is a linear function between kobs and enzyme concentration. If the binding step is fast, the apparent rate constant for the product formation is shown in Equation 3,
k<SUB><UP>obs</UP></SUB>=<FR><NU>k<SUB>2</SUB></NU><DE>1+<FR><NU>K<SUB><UP>d1</UP></SUB></NU><DE>[<UP>E</UP>]<SUB>0</SUB></DE></FR></DE></FR>+k<SUB>−2</SUB> (Eq. 3)
where Kd1 = k-1/k1.

We determined the single-exponential rate constants (kobs) for five different phage clones (Fig. 1) at concentrations of stromelysin ranging from 5-1200 nM (Fig. 4). The reactivity of the clones is widely different; however, without exception, the kobs appears linear to the enzyme concentration throughout the range. Our data clearly agree with Equation 2, suggesting that the slopes of the linear plots should reflect k1, the second-order rate constant for substrate binding to enzyme.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Dependence of the observed first-order rate constants of proteolysis on enzyme concentration. The observed single-exponential rate constants (kobs) were determined at various enzyme concentrations for each of the five substrate phage clones: 10519 (open circle ), 10629 (), 5-2 (), Good (black-square), and 10517 (black-triangle). The kobs values are plotted as a function of stromelysin concentration, and the lines are the linear curve fit to the data. The fit yielded k1, the second-order rate constant for substrate association with enzyme, and k-1, the dissociation rate constant. k1 values are listed in Table I whereas k-1 are 0.011, 0.016, 0.008, 0.052, and 0.012 min-1 for clones 10517, Good, 5-2, 10629, and 10519, respectively.

Recombinant proteins displayed on the tip of bacteriophage particles are not ordinary substrates for enzymes. Phage particles are long cylinders of about 7 nm in diameter and 1-2 µm in length, and each can have five copies of protein pIII (20). Because pIII is present only on one end of the cylinder, there is microscopic heterogeneity in substrate distribution. Interestingly, our data (Figs. 3 and 4) apparently agree with the conventional single-turnover kinetic equations and indicate nothing out of the ordinary on the macroscopic level for the enzymatic reactions involving substrates displayed in multivalencies. We thus conclude that the reaction between an enzyme and its substrate phage obeys the rules of pseudo first-order kinetics. However, we propose that the pseudo first-order kinetic model for substrate phage be regarded as empirical, considering that differences do exist microscopically between homogeneous substrate entities (such as synthetic peptides or purified recombinant proteins) and the recombinant protein substrate generated through phage display.

Application of the Kinetic Model to Inhibitor Study-- We further tested the pseudo first-order kinetic model with clone 10519 by using it to investigate the binding affinity of a competitive inhibitor of stromelysin. A diketopiperazine inhibitor was isolated from a combinatorial synthetic compound library and found to have an IC50 of 9.6 µM against stromelysin.3 In the presence of a competitive inhibitor, the reaction scheme illustrated in Reaction 1 becomes the pathway delineated in Reaction 3.


<AR><R><C><UP>E</UP>+<UP>S</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>−1</SUB></LL><UL>k<SUB>1</SUB></UL></LIM> <UP>E</UP> · <UP>S</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>−2</SUB></LL><UL>k<SUB>2</SUB></UL></LIM> <UP>E</UP> · <UP>P</UP> · <UP>P′</UP></C></R><R><C>K<SUB><UP>i</UP></SUB>⥮+<UP>I</UP></C></R><R><C><UP>EI</UP></C></R></AR>

<UP><SC>Reaction</SC> 3</UP>
In the presence of excess inhibitor, the free enzyme concentration is shown by Equation 4.
[<UP>E</UP>]=<FR><NU>[<UP>E</UP>]<SUB>0</SUB></NU><DE>1+<FR><NU>[<UP>I</UP>]<SUB>0</SUB></NU><DE>K<SUB><UP>i</UP></SUB></DE></FR></DE></FR> (Eq. 4)
Under our assay conditions, the substrate phage titer was on the order of 8 × 109 cfu/ml and the enzyme in the nanomolar range. The presence of inhibitor at 10 times the dissociation constant Ki does not change the pseudo first-order assumption. Thus, Equation 2 can be modified to account for the inhibitor as is shown in Equation 5,
k<SUB><UP>obs</UP></SUB>=k<SUB>1</SUB> <FR><NU>[<UP>E</UP>]<SUB>0</SUB></NU><DE>1+<FR><NU>[<UP>I</UP>]<SUB>0</SUB></NU><DE>K<SUB><UP>i</UP></SUB></DE></FR></DE></FR>+k<SUB>−1</SUB> (Eq. 5)
and the Ki can be determined by varying the inhibitor concentration.

We determined the kobs of the proteolysis of clone 10519 in the presence of varying amounts of the inhibitor. The results, shown in Fig. 5A, indicate that the presence of the inhibitor resulted in a decrease in the rate of proteolysis. Fig. 5B shows the complete plot of the kobs values as a function of inhibitor concentration and the least-squares fit by nonlinear regression to Equation 5. The fit yielded a Ki of 3.8 ± 2.6 µM, which within experimental error is in good agreement with that predicted from the IC50 value.3



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of a competitive inhibitor on the proteolysis of substrate phage. A, the time course of the proteolysis of clone 10519 in the presence of 0 (open circle ), 2.5 (), and 15.6 µM () of the diketopiperazine inhibitor of stromelysin. The enzyme concentration was 40 nM. B, the complete data set plotted as a function of the inhibitor concentration. The line is the least-squares fit of the data to Eq. 5, yielding Ki = 3.8 ± 2.6 µM, k1 = (3.5 ± 0.7) × 104 M-1 s-1 and k-1 = 0.021 ± 0.018 min-1.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown that the reaction of substrate phage with enzyme is a single-turnover event and can be defined by equations of pseudo first-order kinetics. The single exponential rate of product formation reflects the apparent bimolecular binding rate between the enzyme and the substrate.

Several protocols have been successfully applied to the selection of protease substrate phage through screening phage display peptide libraries. The phage particles in the library are either incubated directly with the enzyme in solution (19) or pre-bound to a solid support followed by incubation with the enzyme (13, 21). Based on our finding that the proteolysis reaction is single exponential, the selection processes should enrich phage clones that have shorter half-lives from the proteolysis. A shorter half-life results from a faster rate of substrate association with enzyme, i.e. larger kobs. If it were possible to perform steady-state kinetic experiments with substrate phage, it would imply that substrates with larger kobs would have a smaller Michaelis constant Km and a larger kcat too, if the turnover number was somewhat limited by the binding step. Because it is not feasible experimentally to achieve the conditions of steady-state kinetics with substrate phage, synthetic peptides with the sequence encompassing the entire variable region of the substrate phage have often been used to further characterize the substrates identified in phage display experiments.

The second-order rate constants calculated from the linear plots in our single-turnover studies, shown in Fig. 4, are summarized in Table I along with the steady-state kinetic parameters measured previously for the corresponding synthetic peptides (19). The second order rate constants vary over 100-fold for the five phage clones tested whereas the kcat/Km values differ by 30-fold for the synthetic peptide substrates. It appears that Km, and kcat to some extent, contribute to the increase in the enzyme catalytic efficiency (kcat/Km) of the synthetic peptides. It has been shown in several studies that synthetic peptides, derived from substrate phage clones selected under the same condition, could have very different Km and kcat values (16-18), and furthermore, there were even cases where peptide sequences derived from reactive phage clones failed to show significant reactivity when incorporated into synthetic peptide (19). It can be concluded that overall there is no clear distinction as to which one of the steady-state kinetic parameters is the prevailing contributor to the improvement of catalytic efficiency. Furthermore, there may be no direct correlation between the reactivity of phage clones and synthetic peptides. This may be a consequence of the fact that phage and peptide are two unrelated substrate systems.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Comparison of substrate phage reactivity with that of synthetic peptide substrates

We recognize that recombinant proteins expressed on the tip of a phage particle and synthetic peptides are different molecular entities and as substrates, the substrate phage obeys single-turnover kinetics whereas the synthetic peptides are usually assayed under multiple-turnover conditions obeying steady-state kinetics. This may create difficulties in extrapolating the phage display results to the synthetic peptides. Another consideration for why it is difficult to extrapolate may lie in plausible protein-protein interactions between the enzyme and the bulk of the recombinant pIII protein outside of the substrate region. Several strategies have been employed in designing the flanking sequences for the incorporation of randomized peptides into pIII in the hope of minimizing the effect of the pIII polypeptide outside of the random region (13, 19). It is possible, however, that peptides of certain sequences in the random region could still promote favorable protein-protein interactions between the enzyme and the recombinant pIII, and as a result these clones would be preferentially selected. They may behave poorly as substrate once the substrate sequence is separated from the pIII protein and made into synthetic peptides.

The substrate phage selection procedures rely on capturing and removing unreacted clones from the library, in the hope of enriching reactive ones following each round of selection. That the course of reaction between enzyme and substrate phage is single-exponential (Fig. 3) is thus a key observation. It implies that strong and weak substrates are differentiated by their half-lives of proteolytic cleavage, suggesting that time of incubation and the amount of enzyme are critical factors in designing substrate phage selection experiments. Therefore, few substrates would be found if the selection conditions were too stringent; that is, if the incubation time were too short or the enzyme concentration too low. On the other hand, it would offer little discrimination among good substrates if the conditions were too relaxed.

Stringent conditions are preferred if the population of potential substrates in the library is relatively large. This may apply to enzymes that can tolerate a number of amino acid sequence variations in the substrate. For instance, it has been discovered through practice that stringent conditions were key to the success of substrate phage display experiments with protein tyrosine kinases (14, 15, 25). In general, the selection conditions should be set close to the relaxed scenario when the reactivity of the substrate phage is unknown, and especially when the population of potential substrates in the library is small. This is particularly important for enzymes such as certain proteases that are highly specific in sequence requirements.


    ACKNOWLEDGEMENT

We thank Bruce Mortenson for supplying biotinylated mAB179.


    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.

Dagger Present address: Signature BioScience, Inc., 21124 Cabot Blvd., Hayward, CA 94545.

§ Present address: Zyomyx, Inc., 3911 Trust Way, Hayward, CA 94545.

Present address: Hoffmann-La Roche Inc., 340 Kingsland St., Nutley, NJ 07110.

|| To whom correspondence should be addressed: 3410 Central Expressway, Santa Clara, CA 95051. Tel.: 408-522-5689; and Fax: 408-481-0522; E-mail: danying_cai@affymax.com.

Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M011772200

2 N. A. Sharkov and D. Cai, unpublished results.

3 L. Shi, D. Harris, M. Navre, D. Tien, A. K. Szardenings, and D. A. Campbell, unpublished results. The IC50 value was determined using a thiopeptide substrate whose concentration was estimated to be below the Km of the substrate. For a competitive inhibitor, the IC50 value is always higher than the Ki value.


    ABBREVIATIONS

The abbreviations used are: BSA, bovine serum albumin; cfu, colony-forming unit; CPS, counts per second; HRP, horseradish peroxidase; mAB179, mouse monoclonal antibody 179; ELISA, enzyme-linked immunosorbent assay.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Cwirla, S. E., Peters, E. A., Barrett, R. W., and Dower, W. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6378-6382[Abstract]
2. Devlin, J. J., Panganiban, L. C., and Delvin, P. E. (1990) Science 249, 404-406[Medline] [Order article via Infotrieve]
3. Scott, J. K., and Smith, G. P. (1990) Science 249, 386-390[Medline] [Order article via Infotrieve]
4. Cochran, A. G. (2000) Chem. Biol. 7, R85-R94[CrossRef][Medline] [Order article via Infotrieve]
5. Zwick, M. B., Shen, J., and Scott, J. K. (1998) Curr. Opin. Biotechnol. 9, 427-436[CrossRef][Medline] [Order article via Infotrieve]
6. Souroujon, M. C., and Mochlyrosen, D. (1998) Nat. Biotechnol. 16, 919-924[Medline] [Order article via Infotrieve]
7. Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424[CrossRef][Medline] [Order article via Infotrieve]
8. Kay, B. K., Kurakin, A. V., and Hyde-DeRuyscher, R. (1998) Drug Discovery Today 3, 370-378[CrossRef]
9. Sieber, V., Pluckthun, A., and Schmid, F. X. (1998) Nat. Biotechnol. 16, 955-960[Medline] [Order article via Infotrieve]
10. Ruan, B., Hoskins, J., Wang, L., and Bryan, P. N. (1998) Protein Sci. 7, 2345-2353[Abstract/Free Full Text]
11. Kristensen, P., and Winter, G. (1998) Folding Design 3, 321-328[Medline] [Order article via Infotrieve]
12. Finucane, M. D., Tuna, M., Lees, J. H., and Woolfson, D. N. (1999) Biochemistry 38, 11604-11612[CrossRef][Medline] [Order article via Infotrieve]
13. Matthews, D. J., and Wells, J. A. (1993) Science 260, 1113-1117[Medline] [Order article via Infotrieve]
14. Dente, L., Vetriani, C., Zucconi, A., Pelicci, G., Lanfrancone, L., Pelicci, P. G., and Cesareni, G. (1997) J. Mol. Biol. 269, 694-703[CrossRef][Medline] [Order article via Infotrieve]
15. Schmitz, R., Baumann, G., and Gram, H. (1996) J. Mol. Biol. 260, 664-677[CrossRef][Medline] [Order article via Infotrieve]
16. Coombs, G. S., Bergstrom, R. C., Pellequer, J. L., Baker, S. I., Navre, M., Smith, M. M., Tainer, J. A., Madison, E. L., and Corey, D. R. (1998) Chem. Biol. 5, 475-488[Medline] [Order article via Infotrieve]
17. Ding, L., Coombs, G. S., Strandberg, L., Navre, M., Corey, D. R., and Madison, E. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7627-7631[Abstract]
18. Hervio, L. S., Coombs, G. S., Bergstrom, R. C., Trivedi, K., Corey, D. R., and Madison, E. L. (2000) Chem. Biol. 7, 443-453[CrossRef][Medline] [Order article via Infotrieve]
19. Smith, M. M., Shi, L., and Navre, M. (1995) J. Biol. Chem. 270, 6440-6449[Abstract/Free Full Text]
20. Webster, R. E. (1996) in Phage Display of Peptides and Proteins: A Laboratory Manual (Kay, B. K. , Winter, J. , and McCafferty, J., eds) , pp. 1-20, Academic Press, Inc., San Diego
21. Oboyle, D. R., Pokornowski, K. A., Mccann, P. J., and Weinheimer, S. P. (1997) Virology 236, 338-347[CrossRef][Medline] [Order article via Infotrieve]
22. Harris, J. L., Peterson, E. P., Hudig, D., Thornberry, N. A., and Craik, C. S. (1998) J. Biol. Chem. 273, 27364-27373[Abstract/Free Full Text]
23. Ke, S. H., Coombs, G. S., Tachias, K., Navre, M., Corey, D. R., and Madison, E. L. (1997) J. Biol. Chem. 272, 16603-16609[Abstract/Free Full Text]
24. Matthews, D. J., Goodman, L. J., Gorman, C. M., and Wells, J. A. (1994) Protein Sci. 3, 1197-1205[Abstract/Free Full Text]
25. Gram, H. (1999) Comb. Chem. High Throughput Screen. 2, 19-28[Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.