Site-Directed Chemical Mutations on Abzymes: Large Rate

Subscriber access provided by Northern Illinois University

Article

Site-directed chemical mutations on abzymes: large rate accelerations in the catalysis by exchanging the functionalized small nonprotein components Fumihiro Ishikawa, Masato Shirahashi, Hiroshi Hayakawa, Asako Yamaguchi, Takatsugu Hirokawa, Takeshi Tsumuraya, and Ikuo Fujii ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00574 • Publication Date (Web): 23 Aug 2016 Downloaded from http://pubs.acs.org on August 24, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Site-directed chemical mutations on abzymes: large rate accelerations in the catalysis by exchanging the functionalized small nonprotein components

Fumihiro Ishikawa,a,c Masato Shirahashi,a Hiroshi Hayakawa,a Asako Yamaguchi,a Takatsugu Hirokawa,b Takeshi Tsumuraya,a and Ikuo Fujiia,*

a

Department of Biological Science, Graduate School of Science, Osaka Prefecture

University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan b

Computational Biology Research Center (CBRC), National Institute of Advanced

Industrial Science and Technology (AIST), 2-42 Aomi, Koto-ku, Tokyo 135-0064, Japan

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Taking advantage of antibody molecules to generate tailor-made binding sites, we propose a new class of protein modifications, termed as “site-directed chemical mutation”. In this modification, chemically synthesized catalytic components with a variety of steric and electronic properties can be non-covalently and non-genetically incorporated into specific sites in antibody molecules to induce enzymatic activity. Two catalytic antibodies, 25E2 and 27C1, possess antigen-combining sites which bind catalytic components and act as apoproteins in catalytic reactions. By simply exchanging these components, antibodies 25E2 and 27C1 can catalyze a wide range of chemical transformations including acyl-transfer, β -elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester 1, they showed different catalytic activity. When phenylacetic acid 4 was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of

β-haloketone 2, whereas 27C1 showed no catalytic activity. In this work, we focused on the β-elimination reaction and examined the site-directed chemical mutation of 27C1 to induce activity and elucidate the catalytic mechanism. Molecular models showed that the cationic guanidyl group of ArgH52 in 27C1 makes a hydrogen bond with the P=O oxygen in the hapten. This suggested that during β-elimination, ArgH52 of 27C1 would form a salt bridge with the carboxylate of 4, thus destroying reactivity. Therefore, we utilized site-directed chemical


Page 2 of 41

Page 3 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mutation to change the charge properties of the catalytic components. When amine components 7-10 were used, 27C1 efficiently catalyzed the β -elimination reaction. It is noteworthy that chemical mutation with secondary amine 8 provided extremely high activity, with a rate acceleration [(kcat/Km 2)/kuncat] of 1,000,000. This catalytic activity likely arises from the proximity effect, plus general-base catalysis associated the electrostatic interactions. In 27C1, the cationic guanidyl group of ArgH52 is spatially close to the nitrogen of the amine components. In this microenvironment, the intrinsic pKa of the amine is perturbed and shifts to a lower pKa, which efficiently abstracts the α-proton during the reaction. This mechanism is consistent with the observed kinetic isotope effect (E2 or E1cB mechanism). Thus, site-directed chemical mutation provides a better understanding of enzyme functions and opens new avenues in biocatalyst research.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION Genetic engineering of enzymes provides the means to broaden the scope of chemical transformations and leads to a better understanding of catalytic mechanisms.1-12 Meanwhile, chemists have modified enzymes with unnatural amino acids; examples include “site-specific incorporation of unnatural amino acids”, developed by Schultz. In site-specific incorporation of unnatural amino acids, synthetic unnatural amino acids are incorporated into enzymes by genetic engineering using a chemically acylated suppressor transfer RNA to induce novel steric and electronic properties into proteins.13-16 In the current study, we took advantage of antibody molecules to generate tailor-made binding sites. Using this approach, we propose a new class of protein modification, termed “site-directed chemical mutation”. In this modification strategy, synthetic catalytic components possessing a variety of steric and electronic properties are non-covalently and non-genetically incorporated into specific sites in antibody molecules. This method provides multiple catalytic activities as well as a better understanding of catalytic functions. We recently developed two catalytic antibodies, 25E2 and 27C1, that bear antigen-combining sites to function as apoproteins for binding functionalized small nonprotein components.17,18 By simply changing the functionalized components, antibodies 25E2 and 27C1 catalyze a wide range of chemical transformations including acyl-transfer, β-elimination, aldol, and


Page 4 of 41

Page 5 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


decarboxylation reactions. These antibodies have been elicited by immunization with a haptenic phosphonate diester 1 (Figure 1). The p-nitrophenyl and N-acetylphenyl groups in hapten 1 was designed to elicit substrate- and functionalized component binding sites, respectively, in the antigen-binding site. Although generated using the same antigen, two antibodies showed different catalytic activity. When phenylacetic acid 4 was used as the functionalized component, antibody 25E2 efficiently catalyzed the β-elimination reaction of substrate 2 with a rate enhancement of 2.4×105, whereas 27C1 showed no catalytic activity in this reaction (Figure 1). In this work, using the 27C1-catalyzed β-elimination reaction as a model system, we examined the potential of site-directed chemical mutation. Specifically, we examined which functionalized small components in the active site can be simply replaced to induce new catalytic activity and to improve activity (Figure 2). The results demonstrated that we successfully generated a highly active catalytic antibody, showing a rate acceleration [(kcat/Km)/kuncat] of 106.

RESULTS AND DISCUSSION Cloning and Sequence Analysis of Antibodies 25E2 and 27C1 To understand the observed substrate specificity and catalytic activities of antibodies 25E2 and 27C1, three dimensional computer models of each antigen-combining site were constructed. First, we determined the V-J (light chain) and V-D-J (heavy chain) polypeptide sequences then



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

analyzed the differences (Figure 3C). The cDNAs of the light- and heavy-chain Fab genes were generated from hybridoma mRNA by reverse transcription PCR and then were cloned and sequenced.19 The heavy-chain (Hc) and light-chain (Lc) constant region sequences of 25E2 and 27C1 were found to belong to the IgG1λ subclasses, and the Hc amino acid sequences of 25E2 and 27C1 were classified into the VHIII B subgroup (Figure 3C). Interestingly, despite the single immunization with phosphonate diester hapten 1, the sequences of 25E2 and 27C1 were quite different. In particular, remarkable differences are apparent in complimentary-determining region 3 (CDR3) of the Hc and Lc. Antibody 25E2 has ten amino acid residues in the Hc CDR3 and seventeen amino acid residues in the Hc CDR2. On the other hand, antibody 27C1 has nine and sixteen amino acid residues, respectively. The comparison suggested that the antigen-combining sites of these antibodies would have different shapes (vide infra).

Construction of Three-Dimensional Structural Models of Antibodies 25E2 and 27C1 Model construction was based on the X-ray structures of the frameworks and the CDR loops with the amino acid sequences homologous to antibody 34E4 (PDB code: 1Y0L).20 Thus, the structural coordinates of the most homologous framework regions were used as templates for the model of both antibodies. The six CDR loops, Lc CDR1, Lc CDR2, Lc CDR3, Hc CDR1,


Page 6 of 41

Page 7 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hc CDR2, and Hc CDR3, were modeled by using the X-ray structures (25E2: PDB code: 3CFJ, 1PG7, 2BJM, 1IGC, 1IGT, and 1CFV; 27C1: PDB code: 1A6U, 2ZPK, 1DL7, 2DTM, 3CFB, and 2FBJ) selected according to the canonical rules.21-24 The modeled loops were grafted onto the parent framework structures. The VL/VH interface geometry, followed by hapten docking to the modeled combining site were based on the coordinates of 34E4, an antibody generated against a 2-amino-5, 6-dimethyl-benzimidazole-1-pentanoic acid hapten.25 The modeled complexes of antibodies 25E2 and 27C1 with phosphonate diester hapten 1 are shown in Figures 3A and 3B, respectively. The models show that the hapten is bound in the antigen-combining site via hydrogen bonds and hydrophobic packing interactions. Thus, the p-nitrophenyl moiety of the hapten is buried in a hydrophobic cavity (25E2: TrpH47, PheH100B, TrpL91, and LeuL96, 27C1: TrpH47, TrpL91, and TrpL96). These residues were found to be conserved in the X-ray structures of other catalytic antibodies, CNJ206,26 48G7,27 and 17E828 elicited against aryl phosphonate haptens.29 The observed structural similarity in the hydrophobic pockets is consistent with the concept of “structural convergence”,30,31 and therefore supports the validity of the 25E2 and 27C1 models. Significantly, the model of antibody 27C1 indicates that the guanidyl group of ArgH52, positioned at the edge of the antigen-combining site in antibody 27C1, binds to the oxygen of the P=O bond in the phosphonate hapten via a hydrogen bond. On the other hand, in 25E2, this position is replaced with Ser, which is unable to make an



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

effective hydrogen bond due to the shorter length of the side chain and its more hydrophobic and neutral properties. These differences strongly suggest that the amino acid in the H52 position plays an important role in catalyzing β-elimination reactions. As described above, when phenylacetic acid 4 was used as the functionalized component, antibody 25E2 efficiently catalyzed the β-elimination reaction of substrate 2 with a rate enhancement of 2.4×105. It is likely that an apolar microenvironment in the antigen-combining site increases the reactivity of the carboxylate of 4 through desolvation. On the other hand, antibody 27C1 showed no catalytic activity in this reaction. In 27C1, the ArgH52 appears to make a salt bridge with the carboxylate of 4, destroying the reactivity for proton abstraction in β-elimination. Immunization with the single hapten elicited catalytic antibodies with different microenvironments in the antigen-combining sites. This observation suggested that site-directed chemical mutation would change the microenvironment properties, leading to improved catalytic activity of the antibodies. Therefore, we designed and synthesized a variety of functionalized components to examine the utility of site-directed chemical mutation (Figure 2 and Schemes S1, S2, and S3 in the Supporting Information).

Design and Synthesis of the Functionalized Small Non-Protein Components We first examined the alkyl chain length of the acid components. Benzoic acid and


Page 8 of 41

Page 9 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


phenylpropionic acid derivatives, 5 and 6 possess a different alkyl chain length but share a common antibody-recognition element, N-acetylphenyl moiety. Acid 5 is commercially available and acid 6 was synthesized in one step from commercially available 4-aminohydrocinnamic acid (Scheme S2 in the Supporting Information). Examination of the 3D structural model suggested that changing the functional groups in the non-protein components might improve catalytic properties (Figure 2). In general, site-directed genetic mutagenesis of Asp or Glu with Lys changes not only the functional groups, but also the length of the side chains. On the other hand, site-directed chemical mutation enables replacement of the functional groups while maintaining the length of the side chain. First, to examine the effect of charge, we designed phenethylamine derivative 7 as a functionalized component since amine 7 has the same alkyl chain as acid 4. In addition, we synthesized secondary amine 8 and tertiary amine 9 as outlined in Scheme S1. Primary amine 7 was converted to nosyl protected amine 13.17 Reaction of 13 with excess methyl iodide afforded N-methyl amine 14, which was deprotected by treatment with thiophenol and cesium carbonate to give secondary amine 8. Secondary amine 8 was treated with paraformaldehyde and sodium cyanotrihydroborate to afford tertiary amine 9. To further examine the effect of the alkyl chain length, we synthesized amine components, 10 and 11 (Scheme S1 in the Supporting Information). 4-Aminobenzyl alcohol was converted to



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

diacetyl compound 15, which was selectively deprotected by a transesterification reaction to give alcohol 16. Reaction of 16 with N-bromosuccinimide afforded bromide 17, which was reacted with excess sodium azide to give azide 18. Staudinger reaction of azide 18 gave primary amine 19, which was converted to secondary amine 10 by the same method as that for the synthesis of 8. Synthesis of amine 11 is also described in Scheme S1. 4-Amino-hydrocinnamic acid was reduced to alcohol, an unstable molecule, which was immediately converted to diacetyl compound 22. Diacetyl compound 22 was selectively deprotected by a transesterification reaction to generate alcohol 23. Reaction of 23 with p-toluensulfonyl chloride afforded tosylate 24, which was converted to azide 25. Using the same method as that for amine 10, azide 25 was converted to amine 11. Finally, we synthesized 4-ethylaniline acetamide 12 with no catalytic functional group. Site-directed chemical mutation with 12 is analogous to genetic mutagenesis with alanine. Compound 12 was synthesized in one step from commercially available p-ethylaniline (Scheme S3 in the Supporting Information).

Enzyme Kinetics First, we examined the 27C1-catalyzed β-elimination reaction of 2 using acid components (5 and 6) with different alkyl chain length. The antibody showed no catalytic activity in both cases.


Page 10 of 41

Page 11 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Under these reaction conditions, the guanidium group of ArgH52 in the 27C1 active site would make a salt bridge with the carboxylates of 5 and 6. This electrostatic interaction destroys the reactivity of the functionalized. In natural proteins, such salt bridges most often arise from the anionic carboxylate of Asp or Glu interacting with the ammonium ion of Lys or the guanidium ion of Arg to stabilize a protein conformer.33 Therefore, based on the molecular models described above, we examined site-directed chemical mutation by changing the charge property of individual functionalized components. When amine 7 was used as a functionalized component, antibody 27C1 catalyzed the β-elimination of haloketone 2 in pH 6.0 buffer (10 mM bis-Tris, 100 mM NaCl). Compared to the non-catalyzed reaction, catalysis by 27C1 was highly efficient, showing a rate enhancement [(kcat/Km 2)/kuncat] of 5.3×104; kcat (per binding site) = 3.03 min-1, Km (for 2) = 1.6 mM, Km (for 7) = 3.2 mM, at pH 6.0 (Figure 4 and Table 1). The antibody-catalyzed reaction proceeded in a random, sequential binding manner.34 The antibody-catalyzed rates were measured at fixed concentrations of 7 and varying concentrations of 2. Kinetic parameters were determined by a two-step analysis. First, Lineweaver-Burk (1/V vs. 1/S) plots of the raw data were constructed (Figure 4A). These y-intercepts and slopes were then replotted to yield the actual Vmax and Km (7) values for the 27C1-catalyzed process (Figure 4B). The kcat values were determined from the actual Vmax values. Similarly, the antibody-catalyzed rates were measured at fixed concentrations



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 41

of 2 and varying concentrations of 7. Analogous plots were constructed to give kinetic constants for 2 (Figures 4C and 4D and Table 1). The antibody-catalyzed reaction was competitively inhibited by the addition of hapten 1 or the removal of functionalized molecule 7 (Figure S1 in the Supporting Information). In addition, we examined the chemical mutation of amine 7 with 4-ethylaniline acetamide 12. When replaced with 12, the antibody showed no catalytic activity for the β-elimination of 2. These results showed that amine functionality was essential for the antibody-catalyzed β-elimination. To examine a structural tolerance for functionalized components in antibody 27C1, we next conducted site-directed chemical mutation with the secondary and tertiary amines. When amine 8 was used as a functionalized component, antibody 27C1 showed extremely high activity in the catalysis of β-elimination. Surprisingly, the rate acceleration [(kcat/Km 2)/kuncat] was found to be 1,000,000; kcat (per binding site) = 64.1 min-1, Km (for 2) = 1.9 mM, Km (for 8) = 6.6 mM (Table 1 and Figures S2 and S3 in the Supporting Information). This mutation led to an approximately 20-fold increase in kcat with little effect on Km for substrate 2 and secondary amine 8 in comparison

with

the

27C1-catalyzed

β-elimination

with

primary

amine

7.

The

antibody-catalyzed reaction also proceeded in a random, sequential binding manner.34 On the other hand, when tertiary amine 9 was used as the functionalized component, the antibody showed moderate activity with a rate acceleration [(kcat/Km 2)/kuncat] of 2.6×103; kcat (per binding


Page 13 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


site) = 26.8 min-1, Km (for 2) = 33.5 mM, Km (for 9) = 1.6 mM (Table 1 and Figures S4 and S5 in the Supporting Information). This kinetic analysis showed that the mutation caused a significant increase in Km for 2. Thus, the bulkiness of N, N-dimethyl amine 9 apparently disrupted the conformation of the antigen-combining site and decreased binding affinity for substrate 2. Finally, we examined the effect of the length of the alkyl chains in the amine components. Compared to β-elimination with amine 8, the use of amine 10 with a short alkyl chain (C1) decreased activity, provideing a 64-fold decrease in kcat with little effect on Km for 2 and 10 [(kcat/Km 2)/kuncat] of 6.0×104; kcat (per binding site) = 1.0 min-1, Km (for 2) = 2.2 mM, Km (for 10) = 3.6 mM, at pH 6.0 (Table1 and Figures S6 and S7 in the Supporting Information). In the case of amine 11, which has a longer alkyl chain (C3), no catalytic activity was observed. Thus, as expected, molecular recognition by 27C1 was intolerant of change in the length of the alkyl chains, since the structure of 7 among the amine components tested most closely resembles that of the N-acetylphenyl groups in hapten 1.

Kinetic Isotope Effect Studies Many enzymes catalyze elimination reactions, which involve removal of a proton α to a carbonyl carbon. A recent comprehensive survey suggests that in general these reactions proceed via stepwise ElcB mechanisms involving an enol intermediate.35 In an attempt to



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

compare the antibody-catalyzed elimination reaction to its enzymatic counterparts, kinetic isotope effect studies were carried out using the deuterated substrate 29 (Figure 5A). Substrate 29 was prepared according to the literature procedure (Scheme S4 in the Supporting Information).36 Michaelis-Menten plots were constructed by holding functionalized component 8 at a fixed concentration (1×Km) while varying the concentration of the substrates 2 and 29. The plot of the 27C1-catalyzed elimination of 29 and 8 shows no isotope effect on Km, but does show an effect on kcat, as expected (Figure 5B and Table 2). The kinetic isotope effect on kcat for the antibody-catalyzed reaction is kcatH/kcatD = 4.4. Analogous plots were constructed from data obtained using the functionalized components 7, 9, and 10 (Table 2 and Figures S9, S10, and S11 in the Supporting Information). For the acetate-catalyzed background reaction kH/kD is 3.7. The values of kcatH/kcatD = 3.2 (func. compo. 7), 4.8 (9), and 3.3 (10) for the antibody-catalyzed reaction are consistent with either an E2 or an ElcB transition state. As described above, both antibodies 25E2 and 27C1 were elicited against the phosphonate diester hapten 1. However, the structural and functional characteristics of these antibodies are considerably different. Antibody 25E2 used acid component 4 to efficiently catalyzed the β-elimination reaction of substrate 2, whereas 27C1 showed no catalytic activity for this reaction (Figure 1). Study of the molecular models suggested that the lack of β-elimination activity of 27C1 was due to an electrostatic microenvironment in the antigen-combining site.


Page 14 of 41

Page 15 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


This possibility was examined by site-directed chemical mutations. When amine components 7-10 were individually incorporated into the antigen-combining site, 27C1 efficiently catalyzed the β-elimination reaction. In particular, secondary amine 8 functioned as a general base catalyst to provide extremely high activity. Site-directed chemical mutation with 4-ethylaniline acetamide 12 clarified the essential role of the amine functionality, as this antibody showed no catalytic activity for the β-elimination of 2. In the molecular models, antibody 25E2 possesses a neutral electrostatic environment in the antigen-combining site, whereas antibody 27C1 possesses ArgH52, with the cationic guanidine group at the edge of the antigen-combining site. Antibody 25E2 weakly catalyzed the β-elimination of 2 with amine 7 (kcat/kuncat = 4.9 M) (Figure S8 and Table S1 in the Supporting Information). The weak activity is likely due to proximity effects between the substrate and the amine component. On the other hand, antibody 27C1 efficiently catalyzed the reaction (kcat/kuncat = 82 M), showing 17-fold higher activity than 25E2. The strong activity of 27C1 is due to a combination of proximity effects and electrostatic interactions. In 27C1, the cationic guanidyl group of ArgH52 is spatially close to the nitrogen of amine 7. In the microenvironment of the catalytic site, a pKa of amine 7 is perturbed by electrostatic interactions with the guanidium cation and shifts to lower value than the intrinsic pKa values.37,38 Consequently, the amine component is efficiently deprotonated to act as a general base (Figure 6).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

It is noteworthy that the chemical mutation of primary amine 7 with secondary amine 8 led to an approximately 20-fold increase in kcat, showing extremely high activity with a rate acceleration of 1,000,000. Since primary and secondary amines (ammonium ions) possess similar pKa’s in solution, the increased activity is not due to the basicity of the amines. The efficient general base catalysis of 8 in the 27C1-catalyzed β-elimination presumably reflects an entropic advantage. A steric effect of the N-methyl group in 8 would allow the amine to correctly align with the α-proton of β-haloketone for the β-elimination. These studies are consistent with an E2 or E1cB elimination mechanism, in which the amine components abstract a hydrogen from substrate 2 in the rate-determining step. Natural enzymes display highly sophisticated and complicated microenvironments of the active sites to use a combination of catalytic factors, transition-state stabilization, nucleophilic catalysis, general acid/base catalysis, covalent catalysis, and cofactors, for catalyzing a variety of chemical transformations with large rate accelerations (106–1017). On the other hand, antibody catalysts construct relatively simple catalytic sites. Therefore, insights into the origins of their enormous rate enhancements in enzyme catalysts would be gained by using model systems such as antibody 27C1. Because of its high efficiency and the simplicity of the catalyzed β-elimination reaction, antibody 27C1 presents a well-defined model to investigate the factors that influence general base catalysis at enzyme active sites. In addition, the successful


Page 16 of 41

Page 17 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


incorporation of synthetic catalytic components may facilitate efforts to generate and evolve new active sites to rationally re-engineer existing enzymes or design biocatalysis using synthetic cofactor analogs with novel reactivity.

CONCLUSION In this work, we have reported a new class of chemical modifications with unnatural amino acids for antibody molecules. We term “site-directed chemical mutation”. The chemical mutation described herein facilitated the site-specific substitution of catalytic components with novel steric and electronic properties into abzyme molecules. Molecular models suggested that the ArgH52 in the antigen-combining site of 27C1 plays an important role in catalytic reactivity. Based on these structural insights, site-directed chemical mutations improved catalytic activity to give extremely high active catalytic antibodies. Rate accelerations were improved up to 106 by simply exchanging the functionalized components. These results suggest that a combination of the site-directed chemical mutation with conventional protein engineering, directed evolution39 or site-directed mutagenesis,40 can provide further rate accelerations of catalytic antibodies. Active-site engineering through replacements of functional groups with synthetic small molecules also provides useful insights into enzyme functions, opening new avenues in biocatalyst research.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 41

METHODS Synthesis Detailed synthetic procedures are presented in the Supporting Information.

Antibody Purification Hybridoma cells for antibodies 25E2 and 27C1 were individually grown to 4 L, and the supernatants

were purified by anti-mouse IgG+IgM affinity chromatography (CHROMATOP) (NGK, loaded with

PBS and eluted with 0.2 M Gly-HCl, pH 2.5) to yield purified antibodies. The subclass of each

antibody was determined by using a monoclonal isotyping kit purchased from Amersham (RPN 29).

Cloning and Sequencing of the Antibodies 25E2 and 27C1 Cloning and sequencing of the Fab heavy chain and light chain genes of mAbs 25E2 and 27C1 were carried out after isolation and purification of the messenger RNA from the corresponding secreting cell line using a RNeasy Mini kit (QIAGEN). cDNA was synthesized with a ProSTAR FirstStrand RT-PCR kit (STRATAGENE). Amplification of Fab-H and Fab-L genes was conducted using the following oligonucleotide 25E2: primer pairs: MoVH1 (5’-SAKGTGC AGCTCGAGSAGTCAGGACCT-3’)

and


MoIgG1

(5’-AG

Page 19 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GCTTACTAGTACAATCCCTGGGCACAAT-3’)

for

Fab-H

(5’-AATCTAGTGAGCTCGTTGTGACTCAGGAATCT-3’)

and

gene, Lmd4

Lmd2 (5’-

GCGCGGTCTAGAATTAGGAACACTCAGCACGGGACAA-3’) for Fab-L gene; 27C1: primer pairs: H1 and MoIgG1 for Fab-H gene, Lmd2 and Lmd4 for Fab-L gene.19 DNA fragments resulting from productive amplifications were cloned into pDrive using the PCR Cloning plus Kit (QIAGEN). Multiple Fab-H and Fab-L clones were then sequenced by dideoxy chain termination method using an ABI Prism 3100-Avant genetic analyzer (Applied Biosystems). The amino acid sequences deduced from mAb 25E2 and 27C1 Fab genes are shown in Figure 3C.

Fab Expression Fab expression vector pARAFab was constructed on the basis of the sugar-inducible expression vector pARA7.40,41 All Fab-H and Fab-L gene fragments were inserted into the Xho I - Spe I and Sac I - Xba I restriction sites of pARAFab, respectively. E. coli MC1061 harboring the expression plasmid was cultured in 10 mL expression medium (20 g L-1 tryptone, 10 g L-1 yeast extract, 5 g L-1 NaCl, 2.5 g L-1 K2HPO4) with 100 mg/mL ampicilline at 30 °C until the optical density reached 0.2 at 600 nm. The production of the Fab protein was induced by addition of L-arabinose at final concentration 0.2 % (w/v) and incubating further at 30 °C for 16 h. Culture



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

supernatant was used as sample for ELISA.

Molecular Modeling Computational molecular modeling of antibodies 25E2 and 27C1 with the phosphonate diester hapten 1 involved four steps: search for framework templates, assignment of suitable CDR loops, building the antibody homology model and ligand docking. The antibody template search was performed using template search program of MOE Antibody Modeler (version 2009.10, Chemical Computing Group Inc.) utilizing a combination of two separate search, one for the light and one for the heavy chain template against MOE antibody structure database which was compiled by screening the entire protein collection of Protein Data Bank (PDB)42 for immunogloblins. The framework templates were selected based on sequence similarity to the framework region of 25E2 and 27C1 from the hits with the hapten containing antibody templates. Next step, structural templates for each of the six hypervariable CDR loops were assigned using the sequence and structure information from a representative collection of CDR loop candidate of MOE antibody structure database. The three-dimensional structure of antibodies 25E2 and 27C1 were constructed using the comparative modeling approach incorporated in the builder program of MOE Antibody Modeler. We generated 25 models for a sequence-templates alignment and chose the final model from the ensemble of intermediate 25


Page 20 of 41

Page 21 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


models by using model scoring of the lowest root-mean-square deviation to mean structure. The molecular models of antibodies 25E2 and 27C1 were refined for ligand docking using the Protein Preparation Wizard Script within Maestro (Schrödinger, LLC). Initial coordinates of phosphonate diester hapten 1 was constructed using the Molecular Builder module in MOE. Energy minimization was performed using the OPLS-AA force field in the Conformational Search algorithm in the MacroModel program (Schrödinger, LLC). We performed the flexible docking using Glide ‘Induced Fit Docking (IFD)’ protocol43 (Schrödinger LLC.), where the conformational flexibility of both ligand and the CDR loops of antibodies are modeled by iteratively combining rigid receptor docking (Glide) and protein remodeling by side-chain searching and minimization (Prime) techniques. We generated 50 initial orientations of phosphonate diester hapten 1 in a grid box defined by the reference position of a hapten of the framework templates using the Glide standard precision (SP) mode docking with the soften-potential docking options, which involve scaling the van der Waals radii by 0.25 for antibodies and phosphonate diester hapten 1. In the protein remodeling stage, all residues within a 12.0 Å radius of each initial docked phosphonate diester hapten 1 were refined using Prime. Finally, the best orientation for the phosphonate diester hapten 1 was selected by re-docking and scoring into the refined antibody structure using Glide SP mode.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Enzyme Kinetics The detailed procedures for kinetic assays are presented in the Supporting Information. Representative Kinetic Parameters Determination of β-Elimination Reaction (2 and 7) Assays were performed in a NanoDrop ND-1000 Full-spectrum UV/Vis Spectrophotometer (NanoDrop Technologies, Inc.) at 25 °C in a 10 mM bis-Tris, 100 mM NaCl, pH 6.0, buffer system with 5% (v/v) acetonitrile and 5% (v/v) MeOH as cosolvents. Initial rates were determined spectrophotometically by measuring the absorbance increase at 330 nm as a function of time (2; λmax = 272 nm, 7; λ max = 247 nm, 3; λ max = 312 nm; ∆ε (3-2) (330 nm) = 14,430 M-1 cm-1). The 27C1-catalyzed process was investigated as a random, rapid equilibrium system.34 The antibody-catalyzed rates were measured at fixed concentrations of 7 (0.313 mM ≤ [7] ≤ 2.5 mM) and varying concentrations of 2 (0.5 mM ≤ [2] ≤ 4 mM). Final antibody concentration (10 µM) and temperature (25 °C) were maintained throughout the assay. Kinetic parameters were determined by a two-step analysis. First, Lineweaver-Burk (1/V vs. 1/S) plots of the raw data were constructed. These y-intercepts and slopes were then replotted to yield the actual Vmax and Km (7) values for the 27C1-catalyzed process. The kcat values were determined from the actual Vmax values. Similarly, the antibody-catalyzed rates were measured at fixed concentrations of 2 (0.5 mM ≤ [2] ≤ 4 mM) and varying concentrations of 7 (0.156 mM ≤ [7] ≤ 2.5 mM). Analogous plots were constructed to give kinetic constants for 2. All kinetic assays were


Page 22 of 41

Page 23 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


measured in duplicate. Representative Kinetic Isotope Effect Studies The reactions were carried out at 25 °C in a 10 mM bis-Tris, 100 mM NaCl, pH 6.0 with 5% (v/v) acetonitrile and 5% (v/v) MeOH as cosolvents. Initial rates were determined spectrophotometically by measuring the absorbance increase at λ = 330 nm as a function of time (2; λmax = 272 nm, 8; λ max = 244 nm, 3; λ max = 312 nm; ∆ε (3-2) (330 nm) = 14,430 M-1 cm-1) by a NanoDrop ND-1000 Full-spectrum UV/Vis Spectrophotometer (NanoDrop Technologies, Inc.). A solution of 8 in MeOH was added to an Eppendorf tube, containing the buffer solution with 27C1 (1 µM). The reaction was then initiated by addition of an acetonitrile solution of

β-haloketone 2 or 29 into the mixture. The assays were performed with 1 mM of 8 ( = 1×Km ) and concentrations of 2 or 29 were varied ranging from 250 to 3200 µM. Kinetic parameters [Vmax, kcat, Km (2), Km (29)] were determined by nonlinear least-squares fitting of the initial rate against 2 or 29 concentrations to a hyperbolic curve described by the Michaelis-Menten equation, respectively.

Supporting Information Electronic Supplementary Information (ESI) available: Supporting figures, schemes, and tables; procedures for the synthesis of compounds 6 and 8-12; kinetics assays; full experimental details.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Supporting Information is available free of charge on the ACS Publications website.

Author Information Corresponding Author *E-mail: [email protected] Present Address c

Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and

Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by JSPS KAKENHI Grant Number (20310138). The authors thank H. Hayashi (Osaka Medical College, Japan) for helpful discussions.


Page 24 of 41

Page 25 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References 1. Tarshis, L. C., Proteau, P. J., Kellogg, B. A., Sacchettini, J. C., and Poulter, C. D. (1996) Regulation of product chain length by isoprenyl diphosphate synthases. Proc. Natl. Acad. Sci. USA 93, 15018–15023. 2. Jez, J. M., and Penning, T. M. (1998) Engineering steroid 5β-reductase activity into rat liver 3α-hydroxysteroid dehydrogenase. Biochemistry 37, 9695–9703. 3. van den Heuvel, R. H. H., Fraaije, M. W., Ferrer, M., Mattevi, A., and van Berkel, W. J. H. (2000) Inversion of stereospecificity of vanillyl-alcohol oxidase. Proc. Natl. Acad. Sci. USA 97, 9455–9460. 4. Jürgens, C., Strom, A., Wegener, D., Hettwer, S., Wilmanns, M., and Sterner, R. (2000) Directed evolution of a (βα)8-barrel enzyme to catalyze related reactions in two different metabolic pathways. Proc. Natl. Acad. Sci. USA 97, 9925–9930. 5. Fernandez, S. M. S., Kellogg, B. A., and Poulter, C. D. (2000) Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity. Biochemistry 39, 15316– 15321. 6. Hirooka, K., Ohnuma, S.-I., Koike-Takeshita, A., Koyama, T., and Nishino, T. (2000) Mechanism of product chain length determination for heptaprenyl diphosphate synthase from Bacillus stearothermophilus. Eur. J. Biochem. 267, 4520–4528.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

7. Jez, J. M., Austin, M. B., Ferrer, J.-L., Bowman, M. E., Schröder, J., and Noel, J. P. (2000) Structural control of polyketide formation in plant-specific polyketide synthases. Chem. Biol. 7, 919–930. 8. Schmidt, D. M. Z., Mundorff, E. C., Dojika, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Evolutionary potential of (β/α)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily. Biochemistry 42, 8387–8393. 9. Tanaka, F., Fuller, R., Shim, H., Lerner, R. A., and Barbas, C. F. III (2004) Evolution of aldolase antibodies in vitro: correlation of catalytic activity and reaction-based selection. J. Mol. Biol. 335, 1007–1018. 10. Ijima, Y., Matoishi, K., Terao, Y., Doi, N., Yanagawa, H., and Ohta, H. (2005) Inversion of enantioselectivity of asymmetric biocatalytic decarboxylation by site-directed mutagenesis based on the reaction mechanism. Chem. Commun. 48, 877–879. 11. Toscano, M. D., Müller, M. M., and Hilvert, D. (2007) Enhancing activity and controlling stereoselectivity in a designed PLP-dependent aldolase. Angew. Chem. Int. Ed. 46, 4468–4470. 12. Serafimov, J. M., Gillingham, D., Kuster, S., and Hilvert, D. (2008) The putative Diels-Alderase macrophomate synthase is an efficient aldolase. J. Am. Chem. Soc. 130, 7798– 7799.


Page 26 of 41

Page 27 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., and Schultz, P. G. (1989) A general method for site-specific incorporation of unnatural amino acids into protein. Science 244, 182– 188. 14. Liu, C. C., and Schultz, P. G. (2006) Recombinant expression of selectivity sulfated proteins in Escherichia coli. Nat. Biotechnol. 24, 1436–1440. 15. Hyun, S., and Schultz, P. G. (2008) Biosynthesis of a site-specific DNA cleaving protein. J. Am. Chem. Soc.130, 13194–13195. 16. Lee, H. S., Spraggon, G., Schultz, P. G., and Wang, F. (2009) Genetic incorporation of a metal-ion chelating amino acid into proteins as a biophysical probe. J. Am. Chem. Soc. 131, 2481–2483. 17. Ishikawa, F., Tsumuraya, T., and Fujii, I. (2009) A single antibody catalyzes multiple chemical transformations upon replacement of the functionalized small nonprotein components. J. Am. Chem. Soc. 131, 456–457. 18. Ishikawa, F., Uno, K., Nishikawa, M., Tsumuraya, T., and Fujii, I. (2013) Antibody-catalyzed decarboxylation and aldol reactions using a primary amine molecule as functionalized small nonprotein components. Bioorg. Med. Chem. 21, 7011–7017. 19. Huse, W. D., Sastry, S., Iverson, S. A., Kang, A. S., Alting-Mees, M., Burton, D. R., Benkovic, S. J., and Lerner, R. A. (1989) Generation of a large combinatorial library of the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

immunoglobulin repertoire in phage lambda. Science 8, 1275–1281. 20. Debler, E. W., Ito, S., Seebeck, F. P., Heine, A., and Hilvert, D. (2005) Structural origins of efficient proton abstraction from carbon by a catalytic antibody. Proc. Natl. Acad. Sci. USA 102, 4984–4989. 21. Al-Lazikani, B., Lesk, A. M., and Chothia, C. (1997) Standard conformations for the canonical structures of immunoglobulins. J. Mol. Biol. 273, 927–948. 22. Shirai, H., Kidera, A., and Nakamura, H. (1996) Structural classification of CDR-H3 in antibodies. FEBS Lett. 399, 1–8. 23. Morea, V., Tramontano, A., Rustici, M., Chothia, C., and Lesk, A. M. (1998) Conformations of the third hypervariable region in the VH domain of immunoglobulins. J. Mol. Biol. 275, 269– 294. 24. Oliva, B., Bates, P. A., Querol, E., Aviles, F. X., and Sternberg, M. J. E. (1998) Automated classification of antibody complementary determining region 3 of the heavy chain (H3) loops into canonical forms and its application to protein structure prediction. J. Mol. Biol. 279, 1193– 1210. 25. Thorn, S. N., Danlels, R. G., Auditor, M.-T. M., and Hilvert, D. (1995) Large rate accelerations in antibody catalysis by strategic use of haptenic charge. Nature 373, 228–230. 26. Zemel, R., Schindler, D. G., Tawfik, D. S., Eshhar, Z., and Green, B. S. (1994) Differences


Page 28 of 41

Page 29 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in the biochemical properties of esterolytic antibodies correlate with structural diversity. Mol. Immunol. 31, 127–137. 27. Lesley, S. A., Patten, P. A., and Schultz, P. G. (1993) A genetic approach to the generation of antibodies with enhanced catalytic activities. Proc. Natl. Acad. Sci. USA 90, 1160–1165. 28. Guo, J., Huang, W., and Scanlan, T. S. (1994) Kinetic and mechanistic characterization of an efficient hydrolytic antibody: evidence for the formation of an acyl intermediate. J. Am. Chem. Soc. 116, 6062–6069. 29. MacBeath, G., and Hilvert, D. (1996) Hydrolytic antibodies: variations on a theme. Chem. Biol. 3, 433–445. 30. Charbonnier, J.-B., Golinelli-Pimpaneau, B., Gigant, B., Tawfik, D. S., Chap, R., Schindler, D. G., Kim, S.-H., Green, B. S., Eshhar, Z., and Knossow, M. (1997) Structural convergence in the active sites of a family of catalytic antibodies. Science 275, 1140–1142. 31. Gigant, B., Charbonnier, J.-B., Eshhar, Z., Green, B. S., and Knossow, M. (1997) X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release. Proc. Natl. Acad. Sci. USA 94, 7857–7861. 32. Kabat, E. A., and Wu, T. T. (1991) Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

minigenes, and complementarity-determining regions to binding of antibody-combining sites. J. Immunol. 147, 1709–1719. 33. Anslyn, E. V., and Dougherty, D. A. (2006) Modern physical organic chemistry, Unversity Science Books, Sausalito, California. 34. Segal, I. H. (1975) Enzyme Kinetics: Behabior and Analysis of Rapid Equilibrium and Steady-State Enzyme System, John Wiley and Sons, New York. 35. Gerlt, J. A., and Gassman, P. G. (1992) Understanding enzyme-catalyzed proton abstraction from carbon acids: details of stepwise mechanisms for β-elimination reactions. J. Am. Chem. Soc. 114, 5928–5934. 36. Shokat, K. M., Uno, T., and Schultz, P. G. (1994) Mechanistic studies of antibody-catalyzed elimination reaction. J. Am. Chem. Soc. 116, 2261–2270. 37. Fersht, A. (1999) Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding, W. H. Freeman and Company, New York. 38. Harris, T. K., and Turner, G. J. (2002) Structural basis of perturbed pKa values of catalytic groups in enzyme active sites. IUBMB Life 53, 85–98. 39. Fujii, I., Fukuyama, S., Iwabuchi, Y., and Tanimura, R. (1998) Evolving catalytic antibodies in a phage-displayed combinatorial library. Nat. Biotechnol. 16, 463–467. 40. Miyashita, H., Hara, T., Tanimura, R., Fukuyama, S., Cagnon, C., Kohara, A., and Fujii, I.


Page 30 of 41

Page 31 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(1997) Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization. J. Mol. Biol. 267, 1247–1257. 41. Cagnon, C., Valverde, V., and Masson, J.-M. (1991) A new family of sugar-inducible expression vectors for Escherichia coli. Protein Eng. 4, 843–847. 42. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) The protein data bank. Nucleic Acids Res. 28, 235–242. 43. Sherman, W., Day, T., Jacobson, M. P., Friesner, R. A., and Farid, R. (2006) Novel procedure for modeling ligand/receptor induced fit effects. J. Med. Chem. 49, 534–553.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure Legends

Figure 1. β-elimination reaction catalyzed by antibody 25E2 and the immunized hapten.

Figure 2. Structures of functionalized components with different chemical properties used in the site-directed chemical mutation approach.

Figure 3. Proposed models for hapten binding to models of 25E2 (A) and 27C1 (B) from docking simulations, and sequence alignments of the light and heavy chains of 25E2 and 27C1 (C). Antibody residues within 4 Å of the hapten are shown in stick and line representation (left), respectively. The qualitative electrostatic potential surface of the antibody models (right figure) shows that the amino acid in position 52 of the heavy chain of 27C1, which is close to the N-acetylaminophenyl group of the hapten, is positively charges (Arg); in contrast, the same position in 25E2 is neutral (Ser). All graphics shown in Figures 3A and 3B were generated using PyMOL. Sequence alignment was performed using ClustalW. Asterisks (*), colons (:) and dots (.) represent amino acid residues with identical, strong and weak similarity between 25E2 and 27C1, respectively. Amino acid residues within 4 Å of the hapten are in red font. CDR Loop regions are underlined. Residue numbering and CDRs are defined according to the literature


Page 32 of 41

Page 33 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


procedure.32

Figure 4. Antibody 27C1-catalyzed β -elimination reaction of β -haloketone 2 and amine 8. (A) Lineweaver-Burk plot with 7 held at four fixed concentrations while 2 was varied over concentrations ranging from 0.5 to 4 mM (●[7] = 0.3125 mM; ■[7] = 0.625 mM; ◆[7] = 1.25 mM; ▲[7] = 2.5 mM); v, velocity. (B) Replot of the y-intercepts and slopes of the Lineweaver-Burk plot as a function of [7]-1. (C) An analogous plot was constructed with 2 held at four fixed concentrations while 7 was varied over concentrations ranging from 0.15625 to 2.5 mM (●[2] = 0.5 mM; ■[2] = 1 mM; ◆[2] = 2 mM; ▲[2] = 4 mM). (D) An analogous plot was constructed to provide the kinetic constants for 2. The reactions contained 10 µM antibody 27C1 in assay buffer [10 mM bis-Tris (pH 6.0) and 100 mM NaCl]. The mixtures were incubated at 25 °C. All kinetics assays were measured in duplicate.

Figure 5. Michaelis-Menten plots for the kinetic isotope effect. (A) Structure of the deuterated substrate 29. (B) Michaelis-Menten plots with 8 held at a fixed concentration (1×Km = 1 mM) while 2 and 29 were varied over concentrations ranging from 0.25 to 3.2 mM. The reactions contained 1 µM antibody 27C1 in assay buffer [10 mM bis-Tris (pH 6.0) and 100 mM NaCl]. The mixtures were incubated at 25 °C. All kinetics assays were measured in duplicate.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Molecular interactions for the 27C1-catalyzed β-elimination reaction.

Table 1. Kinetic parameters for the 27C1-catalyzed β-elimination reactions with functionalize components.a a

In the β-elimination reaction with 2 and 4-12, kinetics were measured at 25 °C in assay buffer

[10 mM bis-Tris (pH 6.0) and 100 mM NaCl]. kcat and Km values were calculated from the initial rates using a random, rapid equilibrium mechanism. The bimolecular non-catalyzed rate constants (kuncat) for the β-elimination reactions of 2 and 7-10 were determined using the method of initial rates under identical conditions (7: 3.67 × 10-5 mM-1 min-1, 8: 3.40 × 10-5 mM-1 min-1, 9: 3.09 × 10-4 mM-1 min-1, and 10: 7.28 × 10-5 mM-1 min-1).

Table 2. Comparison of kinetic parameters obtained using β-haloketone 2 and deuterated substrate 29 on 27C1-catalyzed β-elimination reactions.a a

In the β-elimination reaction with 2 or 29 and 7-10, kinetics were measured at 25 °C in 10 mM

bis-Tris, 100 mM NaCl, pH 6.0, 5% (v/v) CH3CN, 5% (v/v) CH3OH or 10% (v/v) CH3CN. The apparent kcat and Km were calculated from initial rates using Michaelis-Menten plots with 7-10 held at a fixed concentration while 2 and 29 were varied.


Page 34 of 41

Page 35 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figures

Figure 1.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2.


Page 36 of 41

Page 37 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4.


Page 38 of 41

Page 39 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6.

Table 1.

Table 2.


Page 40 of 41

Page 41 of 41


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Paragon Plus Environment

Site-Directed Chemical Mutations on Abzymes: Large Rate

Recommend Documents