Enzymes by Design: Chemogenetic Assembly of Transamination

Bioconjugate Chem. 2001, 12, 385−390

385

Enzymes by Design: Chemogenetic Assembly of Transamination Active Sites Containing Lysine Residues for Covalent Catalysis Dietmar Ha¨ring and Mark D. Distefano* Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455. Received September 25, 2000; Revised Manuscript Received February 23, 2001

Artificial enzymes can be created by covalent conjugation of a catalytic active group to a protein scaffold. Here, two transamination catalysts were designed via computer modeling and assembled by chemically conjugating a pyridoxamine moiety within the large cavity of intestinal fatty acid binding protein. Each catalyst included a lysine residue, introduced via site-directed mutagenesis, that promotes catalysis by covalent interactions with the pyridoxamine group. Evidence for such interactions include the formation of a Schiff base with the pyridoxal form of the catalyst and a rate versus pH dependence that is bell shaped; both of these features are manifested in natural transaminases. The resulting constructs operate with high enantioselectivity (83-94% ee) and increase the rate of reaction as much as 4200-fold over the rate in the absence of the protein; this is a modest (12-fold) increase in catalytic efficiency (kcat/KM) compared to the conjugate lacking the lysine residue. Most importantly, these artificial aminotransferases are the first examples of designed bioconjugates capable of covalent catalysis, highlighting the potential of this chemogenetic approach.

Scheme 1. Simplified Transamination Half Reactiona

INTRODUCTION

Artificial enzymes can be designed by covalent conjugation of a catalytic active group to a protein scaffold (Kaiser and Lawrence, 1984; Distefano et al., 1998). This concept has been successfully applied to the construction of a variety of so-called semisynthetic enzymes, such as thiazol-papain (Suckling and Zhu, 1993), flavo-papain (Kaiser and Lawrence, 1984), flavo-hemoglobin (Kokubo et al., 1987), oligonucleotide-nuclease (Corey and Schultz, 1987), or seleno-subtilisin (Wu and Hilvert, 1989; Ha¨ring and Schreier, 1999). The protein scaffold enhances the binding of substrates and provides a chiral environment, thus mimicking an enzymatic active site. However, another important feature of native enzymes has not yet been incorporated in such conjugates: the interaction of functional groups from the protein framework with the cofactor or substrates for covalent catalysis. Pyridoxamine is a versatile catalytic engine for a diverse array of reactions including transamination, decarboxylation, racemization, and aldol condensation. To achieve highly efficient catalysis, interactions with neighboring functional groups are critical. For example, in all known pyridoxamine phosphate utilizing enzymes, the aldehyde form of the cofactor is bound covalently to the -amino group of a lysine residue via a Schiff base linkage. Upon nucleophilic attack of this internal imine by the amino group of an incoming substrate, the lysine residue is released and an internal ketimine complex between the substrate and the aldehyde is formed (Scheme 1). Moreover, this same lysine residue also serves as a general base in the subsequent conversion of the ketimine intermediate to the internal aldimine. Deletion of this residue via mutagenesis results in an enzyme exhibiting a 106 fold decrease in transamination activity (Malashkevich et al., 1995a). * To whom correspondence should be addressed. Phone: (612) 624-0544, Fax: (612) 626-7541, e-mail: [email protected].

a For detailed information about the reaction mechanism and the role of lysine cf. literature (Hayashi et al., 1998; Goldberg and Kirsch, 1996).

To correctly position functional groups in a threedimensional fashion that can modulate catalyst reactivity and selectivity, a sphere-shaped scaffold is useful for the design of an artificial enzyme. Fatty acid binding proteins are a well-characterized family of intracellular transport proteins involved in lipid metabolism (Banaszak et al., 1994; Veerkamp and Maatman, 1995). These proteins bind their cognate ligands in a cavity consisting of a β-barrel and a R-helical lid. Intestinal fatty acid binding protein from rats (IFABP1) is a cysteine-free 15 kDa protein with a internal cavity volume of 600 Å3. Its established structure (Scapin et al., 1992; Hodson et al., 1996) as well as the convenient overexpression of mutants in Escherichia coli make IFABP a conceptually simple framework for the design and assembly of an active site. 1 Abbreviations: AT, aspartate aminotransferase; DTNB, 2,5′dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; EDTA, ethylenediaminetetraaceticacid; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; HPLC, high performance liquid chromatography; IFABP, intestinal fatty acid binding protein; MES, 2-(N-morpholino)ethanesulfonic acid; PMP, pyridoxamine monophosphate; PX, pyridoxamine; TRIS, tris(hydroxymethyl)aminomethane.

10.1021/bc000117c CCC: $20.00 © 2001 American Chemical Society Published on Web 04/27/2001

386 Bioconjugate Chem., Vol. 12, No. 3, 2001

Recently, we developed an approach for catalyst design that combines elements of chemical modification with genetic engineering. For example, a pyridoxamine derivative was covalently linked inside the cavity of IFABP via a disulfide bond to a cysteine residue, which has been introduced by site-directed mutagenesis (Kuang and Distefano, 1998). These experiments demonstrated that the catalytic efficiency of pyridoxamine could be increased by up to 360-fold by introducing the cofactor in the cavity at a specific site.

Here we have used this chemogenetic approach to position functional groups for participation in catalysis in this artificial enzyme. Our goal was to construct a transaminase active site that would contain a lysine residue capable of Schiff base formation and general acid/ base catalysis. An approach based on geometric constraints for residue introduction into an existing catalytic system was developed. Two lysine mutants were prepared and conjugated with pyridoxamine. Spectroscopic evidence for covalent bond formation between the new lysine residues and the cofactor was obtained. The rate versus pH profile for both mutants was measured and shown to be bell shaped, similar to natural transaminases. Finally, kinetic investigation showed that the new lysine mutants exhibited an increased catalytic efficiency relative to the free cofactor and the protein without the lysine residues. MATERIALS AND METHODS

Materials. Pyridoxamine dihydrochloride was converted into 5-(2-pyridyldithio)pyridoxamine 1 as described previously (Kuang et al., 1996). The mutagenic primers were obtained from the Microchemical Facility of the Institute of Human Genetics (University of Minnesota). The site-directed mutagenesis kit (“QuickChange”) was obtained from Stratagene. The expression vector pMON-IFABP was used previously for the preparation of IFABP-V60C (Kuang and Distefano, 1998). The protein purification was carried out at 4 °C unless otherwise noted. Modeling. Molecular modeling experiments were performed using Insight II 97.0/Discover 3.00 (Molecular Simulations). The potential energy was calculated on basis of a consistent valence force field and minimized sequentially by steepest descents and conjugate gradients. The distances and angles of various native aminotransferases used in this study, listed in Table 1, were averaged from literature data (Peisach et al., 1998; Suigo et al., 1995; Miyahara et al., 1994; Malashkevich et al., 1995b, Okamoto et al., 1998). Site-Directed Mutagenesis. Site-directed mutations were introduced in the expression vector of pMONIFABP recombinant plasmid with the QuickChange mutagenesis kit. The sequence for the mutagenic oligomers was 5′-CATGACAACTTGAAAAAGACGATCACACAGGAAG-3′ for L38K and 5′-AAATAAATTCACAGTCAAAAAATCAAGCAACTTC-3′ for E51K. The proteins were overexpressed in E. coli strains JM105, which were grown under shaking at 37 °C in LB media (6 × 1 L) and induced with nalidixic acid. Protein Purification. IFABP-V60C/E51K was purified according to a published procedures (Jiang and

Ha¨ring and Distefano

Frieden, 1993). IFABP-V60C/L38K was expressed as inclusion bodies. They were repeatedly pelleted (3000 g) and resuspended in 0.5 M urea, 50 mM TRIS pH 7.5, 10 mM EDTA, 5 mM dithiothreitol (DTT), and 1% Triton X-100. After a final resuspension in the above buffer without urea, the protein was denatured in 3 M guanidine in buffer A (20 mM HEPES pH 7.5, 10 mM EDTA and 10 mM DTT). Refolding was achieved by slowly dropping the protein solution in a 25-fold volume of 250 mM sodium chloride in buffer A. The IFABP mutant was finally isolated after gel filtration chromatography (Sephacryl S-100). Conjugation to Pyridoxamine Cofactor. Prior to conjugation, the IFABP-mutants were desalted by gel filtration, and the concentration of free thiol was determined by titration with DTNB. The conjugation reagent 5-(2-pyridyldithio)pyridoxamine 1 was added in 10-fold excess, and the reaction was allowed to proceed for 18 h at 20 °C in 20 mM HEPES (pH 7.5). The conjugated protein was purified by gel filtration and the concentration of pyridoxamine determined by its UV absorbance (326 ) 6140 M-1cm-1 in 6 M guanidine, 20 mM HEPES, pH 7.5). To convert the pyridoxamine to the pyridoxal form, the conjugates were incubated with 50 mM R-ketoglutarate for 24 h at 37 °C. The excess of keto acid was removed with a desalting column (Sephadex G25). Kinetic Analysis. Reactions were performed with 50 µM catalyst, 5 mM phenylalanine (in the case of PMP 5 mM tyrosine was used) and varying concentrations of R-ketoglutarate in 0.2 M HEPES (pH 7.5) at 37 °C. The formation of glutamate was monitored by reversed-phase HPLC (Kuang et al., 1996; Buck and Krummen, 1987). Electrospray Mass Spectrometry. For mass spectral experiments, protein samples were desalted (NAP-5 column, elution with 0.5% formic acid), lyophilized, and analyzed by direct infusion (5 µL/min) electrospray ionization mass spectroscopy in water/acetonitrile (1:1) containing 0.5% formic acid at a protein concentration of 50 µM. pH-Dependence. The reactions were performed with 50 µM IFABP-PxK38, 5 mM phenylalanine, and 50 mM R-ketoglutarate acid in “MHP-buffer” (Gloss and Kirsch, 1995) with constant ionic strength I ) 0.2 (25 mM HEPES, 25 mM MES, 50 mM 4-hydroxy-N-methylpiperidine, 0.1 M KCl). The formation of the glutamateenantiomers was monitored by HPLC (Buck and Krummen, 1987), and the data points of the initial rate were fitted to a standard bell curve. RESULTS AND DISCUSSION

Our goal in this work was to design and construct a transaminase active site containing a lysine residue capable of Schiff base formation and general acid/base catalysis. Such a site would be prepared by covalently tethering a pyridoxamine moiety to a cysteine residue (introduced via mutagenesis) within the cavity. On the basis of examination of the crystal structure of IFABP, a V60C mutant form of IFABP was chosen, since it is in a region proximal to the proposed site of entry for fatty acids (and hence putative substrates). Furthermore, attachment of the pyridoxamine to Cys60 positions the cofactor close to the cationic residue Arg126 which could facilitate the binding of carboxylate-bearing substrates via electrostatic interactions (Figure 1). The conformation of the pyridoxamine derivative tethered by a disulfide bond to Cys60 was optimized by energy minimization to provide a starting point for exploring possible sites for Lys introduction.

Chemogenetic Assembly of an Aminotransferase

Bioconjugate Chem., Vol. 12, No. 3, 2001 387

Figure 1. Stereoview of a computational model of IFABP-PxK51 developed from the crystal structure of IFABP (Scapin et al., 1992). The pyridoxal is bound via a disulfide bond to Cys60, and a Schiff base linkage to Lys51; a cationic residue (Arg126) is close to the catalytic center. The figure was generated using MOLSCRIPT (Kraulis, 1991). Table 1. Critical Geometric Parameters for the Placement of a Lysine Residue near Pyridoxamine in a Transamination Catalyst native ATa IFABP-PxK38b IFABP-PxK51b

CR-C4′ (Å)

CR-X (Å)

CR-C5′ (Å)

R (deg)

β (deg)

6.5-8.7 7.1-8.8 6.9-7.6

7.3-8.9 8.0-8.8 5.6-6.4

8.1-9.5 7.4-9.6 6.8-8.1

70-145 134-153 130-140

80-135 33-37 22-35

a The data for native aminotranferases (AT) were averaged from crystal structures of D- and L-aspartate AT as well as aromatic amino acid AT in their pyridoxal, Schiff base, and glutamate ketimine form. b The data for the IFABP conjugates were determined by computational modeling (CR of lysine; X: O-PO32- for native AT or S-Cys60 for IFABP-PX conjugates; R: angle NAr-C4′-CR; β: dihedral angle C5′NAr-C4′-CR).

As guidance for the positioning of a Lys residue capable of Schiff base formation with pyridoxal, we used geometric parameters from the active site of native aminotransferase (AT). Table 1 summarizes critical distances and angles between the cofactor and the CR-atom of lysine in various AT structures. The neighborhood of the IFABPbound pyridoxal was searched with these parameters. This resulted in the identification of 10 possible CRpositions where lysine mutations could be introduced. Four of these mutations were ruled out for reasons of obvious steric hindrance or because they are highly conserved throughout this protein family. Optimized models of the pyridoxal form, the Schiff base to the lysine and the substrate (R-ketoglutarate) ketimine complex were calculated for the other six lysine mutations (L38K, F47K, E51K, Q115K, Y117K, R126K). The geometric parameters of the two most promising mutations, L38K and E51K are summarized in Table 1; a model for one of these constructs is presented in Figure 1. The structural parameters for the L38K and E51K mutants are closely related to those of the native amino transferases that are also included in Table 1 for comparison. It should be noted that although wild-type IFABP has 15 lysine residues, modeling experiments suggest that none of these are oriented appropriately for Schiff base formation with the pyridoxal linked to Cys 60. Based on the modeling results described above, two possible catalysts were assembled by a chemogenetic approach. The desired mutations V60C/L38K and V60C/ E51K of IFABP were introduced by site-directed mutagenesis and the proteins purified to homogeneity. Using the activated pyridoxamine (Px) derivative 5-(2-pyridyldithio)pyridoxamine, the cofactor was chemically linked to Cys60 of both mutants on a 50-100 mg scale with over 90% conjugation efficiency based on DTNB titration and UV/vis spectroscopy of the denatured protein constructs. The resulting assemblies IFABP-PxK38 and IFABPPxK51 (the number indicates the position of the lysine

Table 2. Masses of Various IFABP-Px Conjugates in Their Pyridoxamine Form (amine) and after Reduction with NaBH4 (red. imine) conjugate

calcd mass (Da)

found (Da)

IFABP-Px (amine) IFABP-Px (red. imine) IFABP-PxK38 (amine) IFABP-PxK38 (red. imine) IFABP-PxK51 (amine) IFABP-PxK51 (red. imine)

15179.3 15180.3 15194.3 15177.3 15178.3 15161.3

15178.0 15179.0 15193.0 15188.0 15177.0 15160.0

mutation) show the UV absorption of both the protein (280 nm) and the pyridoxamine (326 nm). Analysis of the proteins via electrospray mass spectrometry clearly indicates the attachment of the cofactor; all of the conjugates are within 2 Da of their expected calculated masses (the pyridoxamine forms) as noted in Table 2. The conjugates IFABP-PxK38 and IFABP-PxK51 were next examined for their ability to catalyze the transamination of R-ketoglutarate and phenylalanine to glutamate and 3-phenylpyruvate.

Kinetic parameters, determined by measuring the initial rate at varying concentrations of R-ketoglutarate, were obtained for IFABP-PxK38 and IFABP-PxK51. The rate data, summarized in Table 3, were compared to the unliganded cofactor as well as to IFABP-Px; this latter construct lacks either of the new lysine residues. Both IFABP-PxK38 and IFABP-PxK51 show improved values for kcat′ and Km′. The overall catalytic efficiency (kcat′/Km′) of IFABP-PxK51 is 4200-fold increased compared to the unliganded pyridoxamine phosphate and 12fold greater compared to IFABP-Px. The predominant kinetic effect of the lysine residues is on Km′. IFABPPxK38 has a Km′ value 2.2-fold lower than IFABP-Px

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Ha¨ring and Distefano

Table 3. Kinetic Parameters for Transaminations Reactions Catalyzed Unbound Pyridoxamine Phospsphate (PMP) and IFABP-Px Conjugates Km′ (mM) kcat′ (h-1) kcat′/Km′ (h-1 mM-1) (kcat′/Km′)rel

PMP

IFABP-Px

IFABP-PxK38

IFABP-PxK51

73 ( 7 3.2 × 10-2 ( 0.2 × 10-2 4.4 × 10-4 ( 0.7 × 10-4 1

1.8 ( 0.5 0.29 ( 0.02 0.16 ( 0.06 360

0.81 ( 0.08 0.44 ( 0.01 0.54 ( 0.07 1200

0.24 ( 0.02 0.44 ( 0.004 1.83 ( 0.17 4200

Figure 2. UV absorption spectra of IFABP conjugates (IFABP-PxK38 dotted line; IFABP-PxK51 dashed; IFABP-Px solid; all 100 µM in 20 mM HEPES, pH 7.5). The insert shows the second derivatives of the spectra.

while IFABP-PxK51 manifests a larger, 7.5-fold decrease. Both conjugates display a small increase in kcat′ (1.5-fold). Molecular modeling suggests that the decreases in Km′ values for the new conjugates are most likely due to the formation of specific interactions between the substrate-derived R-carboxylate group and the -amino groups of the new lysine residues. Finally, it should be noted that the catalytic transamination reactions promoted by IFABP-PxK38 and IFABP-PxK51 are enantioselective, yielding L-glutamate with high enantiomeric purities ranging from 83 to 94% ee depending on the amino acid substrate employed. These levels of enantioselectivity are comparable to those previously obtained with the conjugate IFABP-Px that lacks the engineered lysines. In particular, using R-ketoglutarate and phenylalanine as substrates, glutamate was obtained from IFABP-Px, IFABP-PxK38, and IFABP-PxK51 in 93% ee, 92% ee, and 87% ee, respectively. Using R-ketoglutarate and tyrosine as substrates, glutamate was obtained from the same conjugates in 91% ee, 92% ee, and 92% ee, respectively. Thus, the above kinetic results clearly indicate that the new lysine residues are participating in catalysis and are having a positive influence on catalytic transamination without eroding the enantioselectivity observed with the parent conjugate, IFABPPx. To gain insight into how the introduced lysine residues were interacting with the catalytic center, mechanistic studies were undertaken. IFABP-PxK38 and IFABPPxK51 (both in their pyridoxamine forms) were incubated with excess R-ketoglutarate. Due to the absence of an amino acid substrate, only half of the catalytic cycle can be accomplished, resulting in the conversion of the pyridoxamine cofactor to the corresponding aldehyde. The UV absorbance spectra of these catalyst forms, displayed in Figure 2, showed a band at 422-428 nm, which is characteristic of a pyridoxal-Schiff base complex (Shaltiel and Cortijo, 1970; Metzler et al., 1980). The fluorescence excitation spectra (excitation at 300-500 nm; emission at 510 nm) shown in Figure 3 revealed a corresponding band at 421 nm (Shaltiel and Cortijo, 1970; Metzler et al., 1980). After reduction of the Schiff base to a secondary amine with sodium borohydride, the absorbance and

Figure 3. Fluorescence excitation spectra of IFABP conjugates (ex at 300-500 nm; em at 510 nm; IFABP-PxK38 dotted line; IFABP-PxK51 dashed; IFABP-Px solid; all 10 µM in 20 mM HEPES, pH 7.5). The insert shows the second derivatives of the spectra.

fluorescence bands disappeared. As a control, samples of the IFABP-Px conjugate without the lysine mutation were subjected to the same series of experiments. After reaction with R-ketoglutarate, this conjugate had a maximum in the UV absorption spectrum at 398 nm (Figure 2) and in the fluorescence excitation spectrum at 391 nm (Figure 3), both corresponding to the pyridoxal form (Kallen et al., 1985). Thus, while there are 15 lysine residues present in IFABP-PX, none of these are able to form a Schiff base with the covalently tethered pyridoxal. Presumably, as predicted from modeling experiments, none of these residues have a geometry suitable for Schiff base formation. Only in the case of the lysines introduced by design does Schiff base formation occur. Additional evidence for Schiff base formation between the engineered lysine residues and the pyridoxal cofactor was obtained via electrospray ionization mass spectroscopy; data for these experiments is summarized in Table 2. Analysis of samples of IFABP-PxK38 and IFABPPxK51 following treatment with excess R-ketoglutarate and borohydride revealed a mass loss of 17 m/z compared to the pyridoxamine forms of these conjugates. This is consistent with the loss of ammonia (NH3) that would occur upon intramolecular Schiff base formation with the active site lysine residue. Treatment of IFABP-Px (without a lysine mutation) under similar conditions followed by mass spectrometric analysis resulted in a loss of 1 m/z compared to the amine form, consistent with the simple reduction of the aldehyde to the corresponding alcohol. Finally, in addition to playing a role in Schiff base formation, the engineered lysine residues also appear to participate in catalysis as possible proton donors or acceptors. In Figure 4 the pH dependence of the initial rate of transamination and the enantiomeric ratio of IFABP-PxK38 are shown; similar bell-shaped curves were found in the case of IFABP-PxK51. In contrast, the pH dependence of IFABP-Px (lacking a lysine mutation) catalyzed transamination, measured under the

Chemogenetic Assembly of an Aminotransferase

Figure 4. Top: The initial rate and enantiomeric ratio of the IFABP-PxK38 catalyzed transamination depend on the pH value (pKS1 ) 5.5, pKS2 ) 9.8). IFABP-PxK51 revealed a similar curve with pKS1 ) 7.2 and pKS2 ) 9.8. For comparison, native aspartate aminotransferase shows values of pKS1 ) 6.7-7.2 and pKS2 ) 9.4-10.3 for the pH dependence of kcat in the transamination of R-ketoglutarate and L-aspartate or L-cysteinesulfinate (Gloss and Kirsch, 1995). Bottom: The initial rate and enantiomeric ratio of the IFABP-Px (without a lysine mutation) catalyzed transamination reaction.

same reaction conditions, was linear for both rate and selectivity with a negative slope toward higher pH values. Interestingly, the bell-shaped pH dependence manifested by IFABP-PxK38 and IFABP-PxK51 is similar to that of natural transaminases; clearly the lysine residues introduced into these protein conjugates are influencing catalysis. The plots of the enantiomeric ratio vs pH of IFABP-PxK38 and -PxK51 show a sharp maximum in contrast to that observed with IFABP-Px. Such results suggest that the engineered lysine residues may be directly involved in the protonation of the aldimine intermediate and in determining the enantioselectivity of the reaction. It is helpful to place the results obtained here with the lysine-containing conjugates into context with earlier work using fatty acid binding proteins. In our initial work, a fatty acid binding protein was derivatized with the pyridoxamine using the same reagent as employed here (Kuang et al., 1996). The resulting construct promoted the reductive amination of various R-ketoacids with significant enantioselectivity but at a rate that was actually slower than that mediated by the free cofactor. Subsequent work in which the pyridoxamine was positioned at several sites within the cavity showed that the cofactor was highly sensitive to its location within the protein host (Kuang et al., 1997). IFABP-PX60 was estimated to be at least 9.4 times faster than the original conjugate in a single turnover reaction in which R-ketoglutarate was reductively aminated to glutamic acid and examined after 24 h. These results prompted us to perform a kinetic analysis of the single turnover reaction promoted by IFABP-PX60 as well as to examine the

Bioconjugate Chem., Vol. 12, No. 3, 2001 389

potential for this construct to undergo catalytic turnover in which the net transformation would be the transamination reaction shown in eq 1 (Kuang et al., 1998). Interestingly, it was found that the single turnover reaction was accelerated 62-fold relative to free pyridoxamine when measured at a single substrate concentration (50 mM R-ketoglutarate). Further analysis of the concentration dependence under catalytic conditions enabled values for kcat′ and KM′ to be determined. Comparison of these values with those for the simple pyridoxaminecatalyzed reaction showed a significant decrease in KM′ (41-fold) and a modest increase in kcat′ (9-fold) leading to an overall increase in catalytic efficiency, kcat′/KM′, of 360fold. It should be noted that this rate enhancement in the catalytic reaction occurred in concert with significant enantioselectivity (>90% ee). While the increased catalytic efficiency obtained with IFABP-PX60 was exciting, it is important to emphasize that the role of the protein in this conjugate is limited to serving as a chiral, shapeselective recognition element. In particular, residues that participate in the acid/base chemistry and in modulating the reactivity of cofactor-bound intermediates that would exist in a typical enzyme active site are not present in IFABP-PX60. It was with this in mind that we sought to introduce into the IFABP-PX60 scaffold, lysine residues that could participate in covalent catalysis. The results presented here highlight the successful design and assembly of two catalytic systems based on a protein scaffold that function via covalent catalysis. The interaction of the protein scaffold with the catalytic group was demonstrated by several spectroscopic methods. The introduced lysine residues change the pH profile and the kinetic parameters. The catalytic efficiency increased as much as 4200-fold over that of the free cofactor which is a rate acceleration comparable to that achieved with early examples of catalytic antibodies (Tramontano et al., 1986; Pollack et al., 1986). These semisynthetic aminotransferases are the first examples of bioconjugates designed for covalent catalysis. While the increase in catalytic efficiency due to the lysine residues reported here is only modest, this is not surprising given the absence of additional residues normally present in transaminases that are thought to modulate cofactor/substrate reactivity. Nevertheless, the ability to rationally design catalytic systems that mimic features of enzymatic processes (e.g., covalent catalysis, pH dependence) is a significant achievement. Thus, this approach illustrates the utility of mechanistic information and structural analysis for catalyst design as well as the enormous flexibility that can be harnessed using a combination of chemical and genetic (chemogenetic) methods. ACKNOWLEDGMENT

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