Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
1 To whom correspondence should be addressed. e-mail: hecht{at}princeton.edu
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Abstract |
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Keywords: binary patterning/catalytic activity/combinatorial libraries/esterase activity/protein design
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Introduction |
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For libraries of novel amino acid sequences, the potential for diversity is enormous: for example, a randomly generated combinatorial library of 100 residue sequences would contain 20100 possibilities. This number is so large (20100 > 10130) that a collection containing one molecule of each sequence would fill a volume larger than Avogadros number of universes (Beasley and Hecht, 1997). Thus, the diversity of a random library is more than sufficient for any laboratory screen or selection. However, the quality of such libraries is likely to be quite low. Since most random sequences do not fold into stable protein-like structures (Mandecki, 1990
; Davidson and Sauer, 1994
; Davidson et al., 1995
; Prijambada et al., 1996
), and since well folded structures are a prerequisite for achieving enzymatic activity (Creighton, 1992
; Fersht, 1999
), randomly generated libraries will yield functional proteins only very rarely (Keefe and Szostak, 2001
).
To enhance the likelihood of finding enzyme-like proteins, combinatorial libraries must be confined into those regions of sequence space that are most likely to yield well folded structures. The enormous numerical power of the combinatorial approach must be tempered by elements of rational design.
Over the past decade we have blended combinatorial methods with rational design to produce focused libraries enriched in sequences that fold into stable protein-like structures (Kamtekar et al., 1993; West et al., 1999
; Wang and Hecht, 2002
; Wei et al., 2003a
). Our approach is based on the premise that the binary pattern of polar (O) and non-polar () residues in the linear sequence of a protein can encode a substantial fraction of the information necessary to specify a particular three-dimensional structure. For example, the binary pattern OOOOOOOO, which specifies non-polar residues at every third or fourth position, has a sequence periodicity that matches the
-helical structural periodicity of 3.6 residues per turn. Therefore, de novo sequences constrained by this pattern are predisposed to form amphiphilic
-helices. Conversely, the binary pattern OOOO favors amphiphilic ß-strands, which have an inherent structural periodicity of two residues per repeat. Designed sequences comprising several units of binary patterned amphiphilic secondary structure (
-helices or ß-strands) are expected to fold into globular structures that bury hydrophobic side chains in the protein interior while exposing polar side chains to solvent.
Since a binary pattern stipulates whether a position is polar or non-polar, but does not specify the identities of the side chains, a designed pattern allows for enormous combinatorial diversity. Experimentally, this diversity is incorporated into libraries of de novo proteins, by expressing proteins from libraries of synthetic genes. These genes can be designed to encode libraries of binary patterned amino acid sequences because partitioning of DNA codons into polar and non-polar subgroups is an inherent feature of the genetic code. Thus, the degenerate codon NTN encodes five non-polar amino acids (Met, Leu, Ile, Val and Phe), while the degenerate codon VAN encodes six polar amino acids (Lys, His, Glu, Gln, Asp and Asn) (N represents the DNA bases A, G, C or T, while V represents A, G or C).
We previously reported the design and construction of binary patterned libraries of both -helical and ß-sheet proteins (Kamtekar et al., 1993
; West et al., 1999
; Wang and Hecht, 2002
). Recently, we described a second generation library of sequences designed to fold into four-helix bundles of 102 residues (Wei et al., 2003a
). Biophysical characterization of five proteins from this new library demonstrated that all five were
-helical and stable; and four of the five proteins formed structures that were well ordered and/or native-like (Wei et al., 2003a
). The three-dimensional structure of one of these proteins was determined recently by NMR spectroscopy (Wei et al., 2003b
). As specified by the design, the experimentally determined structure is a four-helix bundle with non-polar side chains buried in the protein interior and polar side chains exposed to solvent. These biophysical and structural studies were performed on proteins chosen arbitrarily from a naïve library that had not been subjected to directed evolution or high throughput screens. Hence, these findings demonstrated that in appropriately designed binary patterned libraries, well folded structures are not rare and may indeed occur frequently.
The finding that binary patterned libraries are rich in well folded structures suggests that such libraries are likely to be far more productive than randomly generated libraries for the discovery of novel proteins with enzyme-like activities. Here we demonstrate that the de novo protein S-824, which has a well ordered four-helix bundle structure (Wei et al., 2003b), has significant esterase activity. The level of activity is similar to, or better than, those of several artificial proteins that were designed explicitly for esterase activity through the use of rational design and computational methods (Broo et al., 1997
, 1998
; Bolon and Mayo, 2001
; Andersson et al., 2002
). Moreover, the level of activity for protein S-824 rivals that observed for some of the first catalytic antibodies, which were isolated by immunological selection (Pollack et al., 1986
; Tramontano et al., 1986
; Tawfik et al., 1990
). To assess whether protein S-824 is a rare fluke or a fairly representative protein in our binary patterned libraries, we also studied the esterase activity of six other proteins from two naïve (unselected) libraries. All six had activities significantly above background, thereby suggesting that appropriately designed binary patterned libraries can provide a rich source of de novo proteins with enzyme-like activities.
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Materials and methods |
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The amino acid sequences of the binary patterned proteins are shown in Figure 1. Proteins were expressed in Escherichia coli and purified by methods described previously (Johnson and Hecht, 1994; Roy and Hecht, 2000
; Wei et al., 2003a
). Stock solutions of protein (12 mM) were made in 50 mM phosphate buffer (pH 7.0). Because p-nitrophenyl esters are poorly soluble in water, stock solutions of substrate were dissolved in acetonitrile, and then diluted into the aqueous reactions. The maximal concentration of acetonitrile in the final reaction mixture was always <0.5%.
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The desired concentrations of substrate and protein were brought to a final volume of 600 µl by adding buffer. The buffers were 50 mM acetate buffer for pH 45.5, 50 mM phosphate for pH 5.87.8, and 50 mM TrisHCl for pH above 8.0. Protein concentration was typically in the range of 20150 µM, and substrate concentration varied from 120 µM to 2 mM. The hydrolysis reaction was followed using a HP 8452A diode array spectrophotometer to monitor absorbance of the product, p-nitrophenol, at 320 nm (at pH <6) or at 400 nm (at pH >6). Absorbance was converted into product concentration by the BeerLambert law. Because the extinction coefficient of the product varies with pH, it was measured in control experiments at each buffer condition.
Data analysis
We wished to compare our results with those reported previously in other systems. Since some of the previous studies analyzed their enzyme-like proteins as simple second-order reactions, while others used the MichaelisMenten model, we calculated rate constants using both models. (Under ideal conditions, where the concentration of substrate is well below the value of KM, kcat/KM would be the same as k2. However, because the concentration of substrate in some of our experiments was close to KM, we calculated rate constants using both models.)
Determination of the second-order rate constant. The observed rate of the reaction () results from the sum of three intrinsic rates: the protein catalyzed reaction, buffer effects and the background uncatalyzed reaction:
= kobs[S]
= k2[E][S] + kbf[bf][S] + kuncat[S]
kobs = k2[E] + kbf[bf] + kuncat
where k2 is the apparent second-order rate constant for catalysis by the enzyme-like protein. The values of kbf and kuncat are readily determined by running the reaction in the absence of protein. Under such conditions:
kobs = kbf[bf] + kuncat
and kbf and kuncat are the slope and y-intercept of a plot of kobs versus [bf]. Once the values of kbf and kuncat are known, then for a given concentration of buffer, the value of (kbf[bf] + kuncat) is a constant. Therefore, in the presence of protein:
kobs = k2[E] + constant
Measurements were made at various concentrations of protein, and the value of k2 was determined as the slope of the line in a plot of kobs versus [E].
MichaelisMenten treatment. The MichaelisMenten model is typically used under conditions where the initial concentration of substrate is much larger than the initial concentration of enzyme (S0 >> E0). Under such conditions the concentration of the enzymesubstrate complex [ES] is small and S0 can be used to approximate [S]. However, for de novo proteins with only modest activity, high concentrations of protein must be used, so E0 cannot be small. Also, as a result of the substrates limited solubility, S0 cannot be large. Therefore, reactions cannot be run with S0 >> E0 so the MichaelisMenten treatment must be used without this simplifying assumption. As described by Broo et al. (1998), this leads to the following relationship:
The rate of the reaction () is measured, and E0 and S0 are known. Km and kcat were determined by plotting
as a function of S0 at a constant value of E0.
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Results |
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The first binary patterned library was designed to encode 74 residue four-helix bundles (Kamtekar et al., 1993). Proteins isolated from this initial library were indeed
-helical. While a few proteins from the original library displayed biophysical properties consistent with native-like structures, most formed fluctuating structures resembling molten globules (Roy et al., 1997
; Rosenbaum et al., 1999
; Roy and Hecht, 2000
). A second generation library was designed recently to test whether elongation of the
-helices would produce a library containing an abundance of well ordered and native-like structures. Helices in proteins from the second generation library were designed to be 50% longer, and the overall length of the sequences was increased from 74 to 102 residues. The second generation sequences were constructed by using binary patterning to elongate the four
-helices of a molten globule-like protein (n86) from the first generation library. Five proteins from this second generation library were subjected to biophysical characterizations, and four of them (all except S-23) were shown to be well ordered and/or native-like (Wei et al., 2003a
). The high-resolution structure of one of these proteins, S-824, was determined recently by NMR spectroscopy, and shown to be a well ordered four-helix bundle (Wei et al., 2003b
). This protein was chosen for the studies described in the current paper.
To assess whether the enzyme-like properties of protein S-824 are unusual or typical of other sequences in our libraries, we chose several other proteins for comparison. These included two proteins from the first generation library, and four from the second generation library. The two proteins from the first generation library are n86, which was shown previously to resemble a molten globule, and M60, which was shown previously to be more native-like (Roy et al., 1997; Rosenbaum et al., 1999
; Roy and Hecht, 2000
). From the second generation library, we chose all five of the proteins (including S-824) that had been characterized previously (Wei et al., 2003a
). These are S-23, S-213, S-285, S-824 and S-836. The sequences of all proteins used in this study are shown in Figure 1.
Neither the first nor the second generation libraries have been subjected to genetic selections or high throughput screens. Thus, all proteins used in this study are from naïve libraries.
Hydrolysis of p-nitrophenyl esters
The reaction monitored in these studies is the hydrolysis of p-nitrophenyl esters, shown below:
This reaction has four distinct advantages: (i) the activation barrier is relatively modest, thereby increasing the likelihood of finding active proteins in a naïve library; (ii) the mechanism is simple and well characterized; (iii) the reaction generates a colored product (p-nitrophenol), thereby facilitating kinetic studies; and (iv) hydrolysis of p-nitrophenyl esters has been used as a model reaction in several earlier studies using very different approaches to search for novel protein-based catalysts (Pollack et al., 1986; Tramontano et al., 1986
; Tawfik et al., 1990
; Broo et al., 1997
, 1998
; Bolon and Mayo, 2001
; Andersson et al., 2002
). This previous history enables direct comparison between our results and those obtained using approaches ranging from computer-aided rational design to immunological selection.
Catalytic activity
For the hydrolysis of p-nitrophenyl acetate, the catalytic activity of protein S-824 is substantially above background (Figure 2). Activity was compared with two controls: 4-methylimidazole is a control for the histidine side chain in isolation without the surrounding protein structure, and sample 48 is a control for residual esterase activity from E.coli that might co-purify with our proteins. [Sequence 48 was part of the original binary code library (Kamtekar et al., 1993), but contains a stop codon early in the gene. A mock protein purification from cells harboring this sequence serves as a control for any background activity from endogenous E.coli proteins.] As shown in Figure 2, the activity of protein S-824 is substantially above these controls.
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For spontaneous (uncatalyzed) ester hydrolysis, the rate of the reaction decreases dramatically with decreased pH. In contrast, for the catalyzed reaction, the effect of pH is relatively modest (<2-fold between pH 7 and pH 8.5; see Figure 3). Hence, the rate enhancement for the catalyzed reaction relative to the uncatalyzed reaction is most striking at lower pH. At pH 7.3, the rate constant for the uncatalyzed hydrolysis of p-nitrophenyl acetate (kuncat) is 3.8x105 min1. At this pH the rate enhancement (kcat/kuncat) by protein S-824 is 8700.
Since hydrolysis of p-nitrophenyl esters by protein S-824 is presumed to involve a histidine nucleophile (see Discussion), it is important to establish that the rate catalyzed by protein S-824 is above that catalyzed by free imidazole. As shown in Figure 2, at pH 7 protein S-824 hydrolyzes p-nitrophenyl acetate 100-fold faster than does 4-methylimidazole.
Activity following multiple turnovers
A defining characteristic of natural enzymes is their ability to catalyze multiple turnovers. To assess whether our de novo protein possesses this enzyme-like property, protein S-824 was incubated overnight with a 10-fold excess of substrate. The protein was then separated from small molecules by filtration and re-assayed. As shown in Figure 4, the multiple turnovers associated with overnight incubation did not reduce the activity of protein S-824.
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Hydrolysis of an ester requires transfer of an acyl group. For enzymes that use a nucleophilic side chain, the mechanism typically occurs in two steps. First, the acyl group is transferred from the substrate to the nucleophilic side chain. Subsequently, the nucleophile is regenerated by transfer of the acyl group to another side chain or to water. To assess whether an acyl group is covalently attached to our de novo protein, S-824 was incubated overnight with a 20-fold excess of the substrate, p-nitrophenyl acetate, and the resulting protein was analyzed by electrospray mass spectrometry. As shown in Figure 5, prolonged incubation with excess substrate caused the attachment of four to seven acetyl groups. Despite multiple acetylations (Figure 5), the protein remains active (Figure 4). This indicates that the active site nucleophile can be regenerated by transfer of the acetyl group to another site on the protein. Moreover, since the protein was incubated with a 20-fold excess of substrate and only four to seven acetyl groups remain attached to the protein, we conclude that acetyl groups are ultimately transferred to water.
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The ability to discriminate between closely related molecules is an essential feature of biological processes. Consequently, many natural enzymes have evolved to possess exquisite substrate specificity. In contrast, our binary patterned proteins, which have neither been designed nor selected to bind particular substrates, would not be expected to possess such specificity. To assess whether protein S-824 can distinguish between closely related substrates, we compared the catalytic rates for the hydrolysis of the p-nitophenyl esters of acetate, propionate and butyrate. As shown in Table I, the values of kcat/kuncat indicate that protein S-824 displays a very slight preference for the propionate ester relative to acetate or butyrate.
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Protein S-824 was chosen for the studies described in the previous sections because it is the first binary patterned protein for which a high-resolution structure is known (Wei et al., 2003b). However, since protein S-824 was not isolated by genetic selections or high throughput screens for activity, our choice to focus on S-824 was somewhat arbitrary. To ascertain whether protein S-824 was simply a lucky choice, or whether its level of activity is representative of activity that might be found frequently in unselected binary patterned libraries, we purified six additional proteins from two binary patterned libraries and measured their esterase activities. The sequences of these proteins are shown in Figure 1.
Surprisingly, all six proteins displayed esterase activity well above background levels. Table II compares the kinetic constants for protein S-824 with the other six binary patterned proteins. Data are presented at pH 8.5, the pH of maximal activity for protein S-824 (see Figure 3).
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(i) If the reaction between enzyme (E) and substrate (S) is modeled as a simple second-order reaction:
then k2 is the apparent second-order rate constant, and the rate of the reaction is k2[E][S]. Values of k2 for the seven proteins are listed in Table II.
(ii) Alternatively, the reaction kinetics can be analyzed according to the MichaelisMenton model:
where ES is the transient enzymesubstrate complex. According to this model, the rate of the overall reaction is kcat[ES]. KM and kcat for the de novo proteins are also listed in Table II.
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Discussion |
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Because the structure of S-824 is known, this protein was chosen for initial studies of esterase activity in an unselected library. As shown in Figure 2, protein S-824 catalyzes ester hydrolysis at rates significantly above background. For the hydrolysis of p-nitrophenyl acetate at pH 7.3 the rate enhancement (kcat/kuncat) by S-824 is 8700.
The maximal activity of S-824 occurs at pH 8.5. The bell-shaped curve of the pH dependence (Figure 3) is consistent with a deprotonated residue acting as a nucleophile and a protonated residue stabilizing a negatively charged intermediate. Because activity drops as pH is lowered from 8.5 to 5.5, and also by analogy with earlier work on novel protein-based esterases (Pollack et al., 1986; Tramontano et al., 1986
; Tawfik et al., 1990
; Broo et al., 1997
, 1998
; Bolon and Mayo, 2001
; Andersson et al., 2002
), we presume the active site nucleophile is histidine.
Natural enzymes, as true catalysts, are not degraded through the course of a reaction, and therefore can catalyze multiple turnovers. As shown in Figure 4, protein S-824 also remains active after many rounds of catalysis. Yet, as shown in Figure 5, the protein is modified by attachment of acyl groups. Acylation was also observed for catalytic antibodies and for rationally designed esterases (Stewart et al., 1994; Broo et al., 1997
; Bolon and Mayo, 2001
). Andersson et al. (2002)
did a systematic study of the acylation of a model protein following reaction with p-nitrophenyl esters. They note that in the first, and rate-limiting step of the reaction, the unprotonated form of the histidine attacks the ester to form an acyl intermediate. The acyl group is then transferred to the flanking lysine in a fast intramolecular reaction and an amide is formed at the lysine side chain... If there is more than one lysine in close proximity to the His residue, the site of modification is determined by intramolecular competition between the flanking lysines (Andersson et al., 2002
). We presume a similar mechanism is responsible for acylation in our system. The sequence of protein S-824 contains 12 histidines and eight lysines. While we do not yet know which of these are responsible for activity, it is clear from the NMR structure (Figure 6) that there are several sites where histidines and lysines occur in close proximity.
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Esterase activity in unselected libraries
As shown in Table II, all seven of the proteins tested thus far catalyze ester hydrolysis at rates significantly above background. Because these proteins were chosen arbitrarilywithout screens or selectionsthese findings suggest that the requirements for this level of activity are relatively promiscuous.
The goal of these initial studies was simply to assess whether esterase activity could be found in focused, but unselected libraries. Although significant activity was observed for all the proteins, these initial experiments are not sufficiently detailed to ascribe a mechanism to the observed activity. Nonetheless, by analogy with earlier work on novel esterases (Pollack et al., 1986; Tramontano et al., 1986
; Tawfik et al., 1990
; Stewart et al., 1994
; Broo et al., 1997
, 1998
; Bolon and Mayo, 2001
; Andersson et al., 2002
), it seems reasonable that at least for some of the proteins the active site nucleophile is histidine. (As mentioned above, the pH profile for the activity of protein S-824 is also consistent with an active site histidine.) Of the seven proteins assayed for activity thus far (Table II), the two proteins from the first generation library (n86 and M60) have five histidines each, while the second generation proteins contain 712 histidines each.
In comparing the activities of the binary patterned proteins to one another (Table II), we note that activity correlates with structural integrity: among the first generation proteins, M60 is more active than n86 (Table II). Earlier studies showed that M60 is also one of the most native-like proteins in the first generation library, whereas n86 is a molten globule (Roy et al., 1997; Rosenbaum et al., 1999
; Roy and Hecht, 2000
). Likewise, for the second generation library, S-23 has the lowest activity (Table II), and is also the only protein from this library that does not possess native-like features (Wei et al., 2003a
). The observed correlation between enzyme-like activity and structural integrity suggests that for the binary code proteins, as for natural enzymes, formation of an active site is favored by formation of a well folded structure (Creighton, 1992
; Matthews et al., 1994
; Fersht, 1999
).
Comparison with other studies
Hydrolysis of p-nitrophenyl esters has been studied by several laboratories as a model reaction for the isolation of novel enzyme-like catalysts. As explained by Menger and Ladika (1987), catalysis of p-nitrophenyl esters is rather easy to achieve. Therefore, the choice of this reaction as a model system has both advantages and disadvantages. The advantage is that the likelihood of finding some level of activity is reasonably good. The associated disadvantage is that although observing activity may be easy, assessing the significance of this activity can be difficult.
Therefore, to evaluate the significance of our results, we compared the activities of our unselected binary patterned proteins to those obtained by four other approaches: (i) computational design of an esterase active site into the natural protein, thioredoxin (Bolon and Mayo, 2001); (ii) iterative rational design of de novo peptides with esterase activity (Broo et al., 1997
, 1998
; Andersson et al., 2002
); (iii) selection of catalytic antibodies from immunological repertoires raised against transition state analogs (Pollack et al., 1986
; Tramontano et al., 1986
; Tawfik et al., 1990
); and (iv) selection of p-nitrophenyl esterases by phage display (Yamauchi et al., 2002
).
Bolon and Mayo (2001) used computational methods to engineer an active site into E.coli thioredoxin. The target activity for their studies was hydrolysis of p-nitrophenyl acetate by a histidine nucleophile. Novel active sites were designed by modeling the transition state of the reaction into numerous loci on the protein, and then computationally searching for mutations and rotamers compatible with formation of an active site. Several active sites, containing two to four mutations, were designed computationally, and the two most promising sequences were synthesized. Both displayed esterase activity above background. At pH 7, the more active of the mutant thioredoxins (called PZD2) displayed a KM of 0.17 mM and a kcat of 0.028 min1 (Bolon and Mayo, 2001
). At a similar pH (7.3), protein S-824 from our binary patterned library had a KM of 3 mM and a kcat of 0.33 min1 (Table III). Thus, PZD2 binds substrate with an apparent affinity (measured by KM) 18-fold better than protein S-824. This is not surprising since the site in PZD2 was computationally designed around a model of the transition state. Unexpectedly, however, the kcat of protein S-824 is 10-fold faster than that of PZD2 (Table III). This is surprising since PZD2 was produced by computational design of an active site into the scaffold of a well folded natural protein, whereas S-824 was chosen arbitrarily from a library of de novo sequences that had neither been designed nor selected for activity.
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In an effort to locate the active site and enhance the substrate selectivity of KO-42, Baltzer and coworkers replaced several of the histidines in the four-helix bundle (Broo et al., 1998). The new peptides had fewer histidineshence fewer sites for activityand saturation kinetics were observed. MichaelisMenten kinetics were reported for a peptide called MNKR, which is similar to KO-42 except that three histidines per chain were mutated. For the hydrolysis of the fumarate ester of p-nitrophenol at pH 5.1, MNKR had a KM of 1 mM and a kcat of 0.01 min1 (Broo et al., 1998
). By comparison, for the hydrolysis of the acetate ester of p-nitrophenol at pH 7.3, protein S-824 has a KM of 3 mM and a kcat of 0.33 min1 (Table III). Although the conditions of the experiments differ somewhat, it seems clear that although S-824 has a weaker affinity for substrate, it catalyzes hydrolysis at a rate similar to (or perhaps faster than) the rationally designed MNKR.
A third approach for isolating novel esterases involves selecting catalytic antibodies. The first catalytic antibodies were reported in 1986 (Pollack et al., 1986; Tramontano et al., 1986
). Schultz and coworkers isolated MOPC167, an antibody that catalyzed hydrolysis of p-nitrophenyl carbonate with a KM of 0.21 mM and a kcat of 0.4 min1 (Pollack et al., 1986
). Concurrently, Lerner and coworkers isolated 6D4, which catalyzed ester hydrolysis. Depending on the substrate, 6D4 had a KM of 1.9 or 0.62 µM, and a kcat of 1.6 or 0.5 min1 (Tramontano et al., 1986
). In 1990, Tawfik et al. (1990)
reported several antibodies that catalyzed the hydrolysis of p-nitrophenyl esters with KM values ranging from 0.06 to
30 mM, kcat values ranging from 0.63 to 2.39 min1, and enhancement ratios (kcat/kuncat) ranging from 2600 to 9700. These early antibodies functioned by stabilizing the transition state corresponding to direct hydroxide attack on the scissile carbonyl (Hilvert, 2000
). In contrast, our proteins appear to catalyze hydrolysis by promoting acyl transfer to histidine, followed by transfer to water. Despite these different mechanisms, it is instructive to compare the kinetic parameters reported for the early catalytic antibodies to those of the binary patterned protein S-824 (Table III). The catalytic antibodies typically bind substrate with higher affinities than protein S-824. It would be surprising if this were not the case, since the antibodies were selected by the immune system for binding to transition state analogs, whereas S-824 is from an unselected library. The catalytic antibodies also exhibit greater substrate specificity (Hilvert, 2000
) than observed for protein S-824 (Table I). This is also not surprising since the antibodies were raised against transition state analogs. It is surprising, however, that the catalytic rate (kcat) and rate enhancement (kcat/kuncat) of protein S-824 are in the same range as many of the early catalytic antibodies (Table III).
As an alternative to the catalytic antibodies generated by the mammalian immune system, one can also use phage display to select among diverse sequences for those that bind transition state analogs. Yamauchi et al. (2002) displayed random polypeptides of
140 residues on the surface of phage, and selected for binding to a transition state analog for the p-nitrophenyl esterase reaction. In contrast to typical phage display experiments, Yamauchi et al. (2002)
used very small libraries containing only 10 clones per generation. In each generation, the clone showing the highest affinity for the target was subjected to random mutagenesis, and advanced to the next round. After six generations, the sequence with the highest esterase activity, YSLP6-1, had a kcat/KM at pH 5 of 0.064 mM1 min1. (Individual kcat and KM values were not reported.) By comparison, after no rounds of selection, protein S-824 showed a kcat/KM at pH 7.3 of 0.11 mM1 min1 (Table III).
In summary, when comparing the binary patterned proteins to the artificial protein-based esterases described in the previous four examples, it is important to note that the binary patterned proteins (i) were constructed entirely de novo and not engineered onto a pre-existing natural protein scaffolds, (ii) were not designed explicitly to bind substrate, and (iii) were chosen at random and not by selections or high throughput screens. Nonetheless, these proteins possess enzyme-like activity, which although lower than those of natural enzymes, rivals those of earlier artificial esterases devised using natural protein scaffolds, rational design, immunological selection or phage display.
These findings suggest that although achieving the exquisite levels of activity and specificity typical of natural enzymes may have required eons of evolutionary selection, moderately active macromolecular catalysts (Hollfelder et al., 1996; Suh and Oh, 2000
) may occur frequently in focused combinatorial libraries that have undergone neither evolutionary selection nor active site design.
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Acknowledgements |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beasley,J.R. and Hecht,M.H. (1997) J. Biol. Chem., 272, 20312034.
Bolon,D.N. and Mayo,S.L. (2001) Proc. Natl Acad. Sci. USA, 98, 1427414279.
Broo,K.S., Brive,L., Ahlberg,P. and Baltzer,L. (1997) J. Am. Chem. Soc., 119, 1136211372.[CrossRef][ISI]
Broo,K.S., Nilsson,H., Nilsson,J. and Baltzer,L. (1998) J. Am. Chem. Soc., 120, 1028710295.[CrossRef][ISI]
Chen,G., Hayhurst,A. Thomas,J.G., Harvey,B.R., Iverson,B.L. and Georgiou,G. (2001) Nat. Biotechnol., 19, 537542.[CrossRef][ISI][Medline]
Creighton,T.E. (1992) Proteins: Structures and Molecular Properties, 2nd edn. W.H. Freeman, New York.
Davidson,A.R. and Sauer,R.T. (1994) Proc. Natl Acad. Sci. USA, 91, 21462150.[Abstract]
Davidson,A.R., Lumb,K.J. and Sauer,R.T. (1995) Nat. Struct. Biol., 2, 856864.[ISI][Medline]
Fersht,A. (1999) Structure and Mechanism in Protein Science. W.H. Freeman, New York.
Hanes,J. and Plückthun,A. (1997) Proc. Natl Acad. Sci. USA, 94, 49374942.
Hilvert,D. (2000) Annu. Rev. Biochem., 69, 751793.[CrossRef][ISI][Medline]
Hollfelder,F., Kirby,A.J. and Tawfik,D.S. (1996) Nature, 383, 6063.[CrossRef][ISI][Medline]
Johnson,B.H. and Hecht,M.H. (1994) Biotechnology, 12, 13571360.[ISI][Medline]
Kamtekar,S., Schiffer,J.M., Xiong,H., Babik,J.M. and Hecht,M.H. (1993) Science, 262, 16801685.[ISI][Medline]
Keefe,A.D. and Szostak,J.W. (2001) Nature, 410, 715718.[CrossRef][ISI][Medline]
Mandecki,W. (1990) Protein Eng., 3, 221226.[Abstract]
Matthews,B.W., Craik,C.S. and Neurath,H. (1994) Proc. Natl Acad. Sci. USA, 91, 41034105.
Menger,F.M. and Ladika,M. (1987) J. Am. Chem. Soc., 109, 31453146.[ISI]
Pollack,S.J., Jacobs,J.W. and Schultz,P.G. (1986) Science, 234, 15701573.[ISI][Medline]
Prijambada,I.D., Yomo,T., Tanaka,F., Kawama, T, Yamamoto,K., Hasegawa,A., Shima,Y., Negoro,S. and Urabe,I. (1996) FEBS Lett., 382, 2125.[CrossRef][ISI][Medline]
Rosenbaum,D.M., Roy,S. and Hecht,M.H. (1999) J. Am. Chem. Soc., 121, 95099513.[CrossRef][ISI]
Roy,S. and Hecht,M.H. (2000) Biochemistry, 39, 46034607.[CrossRef][ISI][Medline]
Roy,S., Ratnaswamy,G., Boice,J.A., Fairman,R., McLendon,G. and Hecht,M.H. (1997) J. Am. Chem. Soc., 119, 53025306.[CrossRef][ISI]
Smith,G.P. (1985) Science, 228, 13151317.[ISI][Medline]
Stewart,J.D., Krebs,J.F., Siuzdak,G., Berdis,A.J., Smithrud,D.B. and Benkovic,S.J. (1994) Proc. Natl Acad. Sci. USA, 91, 74047409.[Abstract]
Suh,J. and Oh,S. (2000) J. Org. Chem., 65, 75347754.[CrossRef][ISI][Medline]
Tawfik,D.S., Zemel,R.R., Arad-Yellin,R., Green,B.S. and Eshhar,Z. (1990) Biochemistry, 29, 99169921.[ISI][Medline]
Tramontano,A., Janda,K.D. and Lerner,R.A. (1986) Science, 234, 15661570.[ISI][Medline]
Wang,W. and Hecht,M.H. (2002) Proc. Natl Acad. Sci. USA, 99, 27602765.
Wei,Y., Liu,T., Sazinsky,S.L., Moffet,D.A., Pelczer,I. and Hecht,M.H. (2003a) Protein Sci., 12, 92102.
Wei,Y., Kim,S., Fela,D., Baum,J. and Hecht,M.H. (2003b) Proc. Natl Acad. Sci. USA, 100, 1327013273.
West,M.W., Wang,W., Patterson,J., Mancias,J.D., Beasley,J.R. and Hecht,M.H. (1999) Proc. Natl Acad. Sci. USA, 96, 1121111216.
Yamauchi,A., Nakashima,T., Tokuriki,N., Hosokawa,M., Nogami,H., Arioka,S., Urabe,I. and Yomo,T. (2002) Protein Eng., 15, 619626.
Received September 8, 2003; accepted October 20, 2003 Edited by Greg Winter