The state of antibody catalysis

459

The state of antibody catalysis David B Smithrud* One of the fascinations possibility

mechanistically

and the immunological more thoroughly screening

and applying

encountered

repertoire

antibodies

the mechanisms

transformation

realm of chemistry the catalytic include

of catalytic

of harnessing

for chemical

and Stephen J Benkovict

in organic

of antibodies

to enzymes

them to the broad synthesis.

Recently,

has been extended

more complex response

Abzymes versus enzymes

is the

available

bimolecular

to the hapten

to

reactions

can be

examined as a result of the advent of new

technology

using bacterial

phages or auxotrophic

cell lines.

Addresses Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA *e-mail: dbs5@psuedu te-mail: sjbl @psu.edu Current Opinion in Biotechnology 1997, 8:459-466 http://biomednet/elecref/0958166900800459 0 Current Biology Ltd ISSN 0958-l 669 Abbreviation BPTI bovine pancreatic trypsin inhibitor

Introduction The successful conversion of antibodies to catalysts [l-13,14’] can be attributed to the diversity of the combining site, allowing for recognition and chemical modification of a diverse set of substrate molecules. Catalytic antibodies, or abzymes, are generally successful in reactions with modest activation barriers with the caveat that the comparison of catalytic efficiency (defined, for example, by the ratio of R,,t/K,) with the rate measured for the uncatalyzed reaction is often misleading. Such parameters are not appropriate for determining actual product yields if the antibody cannot be saturated under the reaction conditions or is inhibited by the product. A more direct measure of efficiency is product flux, in other words, how much product is actually obtained within a reasonable time. Even so, the initial results of antibody catalysis have been quite satisfying, but the question now is whether abzymes can be created that are capable of promoting reactions requiring extensive acid-base catalysis and with reaction flux competitive with other methods. The purpose of this review is to stimulate future improvements in abzymes by giving a cursory comparison of their structural and catalytical characteristics with those of enzymes, as well as by a critical examination of their successes and failures, and by highlighting screening technology that may ultimately determine the potential of antibodies to act as catalysts.

It is reasonable to compare the physical and chemical properties of abzymes with enzymes because both proteins process their substrates through their respective protein-substrate complexes. Behavioral differences can possibly be gleaned by examining their physical properties. Antigen binding occurs in the variable domains of antibodies through six loops associated with the heavy and light chains. The combining sites are either small pockets or elongated grooves on the antibody surface, depending on the size of antigen. Small aromatic molecules generally used for haptens are bound in pockets, but are not completely buried within the antibody. For example, the tight binding of progesterone-l la-ol-hemisuccinate to antibody DB3 is accomplished with only 81% of the antigen being buried [15]. DB3 is crossreactive with steroids other than progesterone-l la-01-hemisuccinate, and even though they are buried to a greater extent, these steroids are less tightly bound. Haptens that contain the common nitrophenyl epitope have been observed by X-ray analysis to be buried to the extent of 68% [16] to 90% [17]. One reason for incomplete enclosure of haptens is the linker used to attach the hapten to the carrier protein lengthens the antigen. An open combining site will expose part of the substrate, which removes the opportunity to utilize, either through recognition or chemical bond formation, that face of the substrate. In enzymology, the importance of enclosing the substrate within the active site is debated, but it is known that enzymes, in general, completely bury their substrates or, in some cases, perform cavity closure via loop motion. For example, covering the active site of dihydrofolate reductase by loop I is important for the catalysis of hydride transfer between NADPH and the pterin ring of dihydrofolate [18]. Mutations in loop I that reduce its ability to cover the active site increase the release rate of folate product from 1 s-l to 8s-1 but effect a SOO-fold reduction in the rate of hydride transfer. A similar response occurs in triosephosphate isomerase, which catalyzes the interconversion of D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate [19]. A mobile loop on the enzyme comes into contact with the phosphate group of the substrate, and mutations in this loop reduce the isomerization rate nearly 105-fold. Molecular mechanics calculations suggest that this reduction in the catalytic rate is due, in part, to the exposure of the phosphate to the solvent and to the loss of enzyme rigidity. The triosephosphate isomerase and dihydrofolate reductase examples imply that controlled motion of the protein surface may be necessary for highly efficient catalysis to take place. A full range of theories exists

460

Protein engineering

on the importance of conformational motion in catalysis by enzymes. One features a rigid ‘lock and key’ active site, another suggests ‘induced fit’ by ligand binding, and finally a third highlights a highly ‘mobile rack’ mechanism capable of substrate distortion. Similar criteria exist for antigen binding, where both lock and key [20] and induced fit [21] models have been used to describe the extent of conformational changes accompanying antigen binding. The examination of thermodynamic values for binding and catalysis may provide insight as to whether enzymes and abzymes subdivide in the same manner the free energy changes that occur as substrate is converted into product. Binding interactions can be dramatically different between antibodies and enzymes. For example, the binding of the Fv fragment of the monoclonal antibody D1.3 with lysozyme is enthalpitally driven AC =-1 1.4 kcal mol-1, M = -20.3 kcal mol -1,

TM z-S.9 kcal mol-1 [22]. In contrast, binding of alpha chymotrypsin to the peptide inhibitor BPTI is driven by a favorable entropic change AC--lo.7 kcal mol-1, AH= 2.5 kcal mol-1, TAJ’= 13.2 kcal mol-1 [23]. One interpretation of the thermodynamic values for binding is that the antibody pocket is fairly rigid, resulting in an enthalpically driven process. Although there is a paucity of studies on the affect of temperature on catalysis, it appears that abzyme catalysis is also dominated by an enthalpic term. For example, comparisons of the temperature effect on the catalyzed and uncatalyzed rates for the isomerization of the bridged biphenyl structure 1 (Figure 1) [24] and for the sigmatropic reaction of the N-oxide structure 3 (Figure 1) [25] reveal that catalysis stems from a reduction in AH* (Table 1). This lowering of AH+ is also seen in the background reaction upon changing solvent from water to DMF, suggesting that the polarity of the antibody pocket plays a dominating role.

(a)

(b) BH 02N b

N-

-HNO

0

NH(CH&CQH

CN O-

8

*r

Some recent reactions that have been catalyzed by antibodies raised against the appropriate hapten (shown below the reaction) where the bracketed species is the putative transition state structure. (a) lsomerization of 1 tested for the rate enhancement gained by transition state stabilization, and the elimination reaction of 3 probed for the rate enhancement gained by ground state destabilization caused by burying the charged substrate into an apolar combining site. (b) Another example of ground state destabilization catalysis but, in this case catalysis arose from the flattening of the aromatic rings of 6, and finally, antibody catalysis of the deprotonation of benzisoxazole (structure 6) by a catalytically active, sidechain residue B: induced within the combining site using ‘bait and switch’ technology.

The state of antibody

catalysis

Smithrud and Benkovic

461

Figure 2

(a)

#r

AcHN0

WACGH~)‘P

=Ph

0 ~(CsH4W’Q

10

11

13

H2N 14 (c) 0

w NH2

15

(cH~)~H 0

WNk

(CH2j2eH 0

16A Studies on bimolecular reactions catalyzed by antibodies raised against the haptens indicated below the arrows. (a) The dipeptide derivatives 10 and 11 were coupled by an antibody ligase raised against hapten 12. (b) The retro-aldol reaction of 13 was catalyzed by an antibody induced to promote aldol condensation. (c) The reaction selected to investigate heterologous immunization which attempts to induce two active site residues within a combining site through immunization with the haptens 16A and 16B.

Because recognition it is reasonable that antibodies a simpler, would be fashioned

and binding is easier than catalysis, in light of the normal function of more rigid cavity with a common fold for binding. Schultz and co-workers

[26] have examined the maturation of esterase antibodies by studying germline and mature antibodies raised to a phosphonate hapten. The flexibility of the loops in the variable domains allows for a variety of antigen binding

462

Protein engineering

Table 1 Comparison

of the temperature

effect on catalyzed

and uncatalyzed

reaction

Catalyzed Structure 1 3

Reaction lsomerization Sigmatropic

Enthalphy (Am) 23.5 kcallmol 27.2 kcallmol

Entropy (As) -0.43 cal/(mol K) -0.29 cal/(mol K)

modes for germline antibodies. Maturation results in the selection of the optimal binding mode, which results in stronger binding of the hapten (Ktlmarure=0.005pM better catalysts and Kd,germline = 135 pM) and consequently and Rear/Km,germline (kcaJKm,mature = 1.4 x 104 min-1 M-r = 1.7~ lOzmin-1 M-1. As the antibody matures, the geometry of the binding pocket changes as a result of somatic mutations in the amino acids within the framework regions that do not make contact with the hapten. This suggests that the amino acids that do make contact with the hapten are now locked into a conformation that maximizes binding.

Available

mechanisms

If catalytic antibodies conform more to a lock and key hypothesis for the catalytic operation of their active sites, it is fortunate that binding is the raison d’etre of antibodies. The technological goal is to synthesize a perfect transition state analog in order to elicit an antibody capable of providing a maximum difference in the energy between substrate and hapten binding and converting this binding energy into a catalytic turnover of substrate. This is a lofty goal because transition states by definition cannot be synthesized and it may also be impossible to create a hapten that represents the entire reaction coordinate. It appears that the maximum flux for transition state (TS) stabilization catalysis is restricted to be on the order of 105, which is calculated by using the equation for determining the maximum flux ratio for catalysis [27*,28], an abzyme concentration of (Vcat/vnon)max = [Ab]/KTs, lO-SM, and a general value for KTS of lo-loM (where of binding energy into KTS =Kd,hapten ). The conversion turnover is generally accomplished by overcoming the translational and rotational modes of freedom of the substrate by confinement within the antibody’s combining site and by specific interactions through noncovalent bond formation between the substrate and sidechains of the amino acids. Greater catalytic rates could potentially occur for abzymes that perform covalent catalysis. There are, however, only a few known examples of abzymes that lower the activation barrier via covalent bond formation between an active site residue and substrate, owing to the stringent requirements for reactive group alignment. Accordingly, the more suitable reactions for abzyme catalysis should be ones that only require lowering the energy of activation through noncovalent interactions, and the two reactions discussed previously are good examples. In the isomerization of the bridge biphenyl (structure 1) [24]

rates. Uncatalyzed Enthalphy (Ati) Entropy (ASi) 26.5 kcal/mol 30.6 kcallmol

0.43 cal/(mol K) -2.6 cal/(mol K)

Reference 1241 [251

the reaction should proceed through a planar transition state which is represented by the coplanar transition state analog (structure 2). The majority of antibodies capable of catalyzing this reaction, however, did not fully convert excess binding energy (Km/Kd,hapten = 2000-3000) to catalysis (Rcar/kuncat = ‘200-2700). In the catalysis of the pericyclic reaction of the N-oxide (structure 3) to give 4-methoxystyrene (structure 4) [25], the activation barrier was lowered mainly through the relief of ground state destabilization, the result of burying the charged substrate in an apolar pocket. The cetrahydrofuran hapten (structure 5) was used to raise an antibody that contained an apolar binding pocket necessary to favor the loss of charge on the substrate once it is bound. This reaction was also enhanced by a factor of 103, which is consistent with a general value for constrained, intramolecular model systems for small molecules [29]. One method of programming the antibody-binding pocket to provide active general acid-base catalysis is to induce, through hapten design, the creation of specific interactions in the combining site [30]. The haptens are not necessarily transition state analogs but usually contain a polar group that will be countered by an appropriate complementary residue in the binding pocket that will be catalytically active, giving rise to the term ‘bait and switch’. For example, using a methylated pyridinium hapten (structure 7) antibodies were sought that could activate the prodrug (structure 6) using a basic residue to catalyze a reverse Diels-Alder reaction by releasing HNO (which can subsequently be oxidised to nitric oxide by superoxide dismutase) [31]. Placement of an appropriately positioned sidechain to facilitate acid-base catalyzed cleavage was not achieved. Although, catalytic antibodies were obtained (L&ncat = ZOO), the activation barrier was lowered as a result of relief of ground state destabilization stemming from flattening the phenyl rings of the substrate to fit into the planar binding pockets. Introducing an appropriate sidechain by hapten design was similarly unsuccessful in an antibody capable of catalyzing the hydrolysis of Paraoxon to cure insecticide poisoning [32]. A successful example of the bait and switch technology is the base-promoted opening of the benzisoxazole (structure. 8) catalyzed by antibody 34E4 with a rate enhancement greater than 108 (this value does not compensate for the pKa difference of the general base in the two reactions; k,JK,,, =5.5x 103 M-1 s-1 and R~eo- = 5.3 x 10-S M-1 s-1) [33]. (It should be noted that bovine serum albumin provides a similar rate enhancement for the same substrate by using a fortuitous catalytic pocket [34].) A carboxylic

The state of antibody catalysis Smithrud and Benkovic

acid sidechain was programmed into the combining site by using the benzimidazole hapten (structure 9). Finding the source for this rate enhancement is complex but a principal component is the sensitivity of the deprotonation reaction to the low polarity of the combining pocket [33]. The more complex bimolecular reactions have been catalyzed by constructing combining sites capable of recognizing and activating two substrates. For example, the antibody 16G3 raised against hapten 12 was capable of catalyzing the bimolecular coupling of two peptide derivatives, for example, structures 10 and 11 (Figure Za) with yields up to 70% [35]. The phosphonate and aromatic moieties in 12 induced pockets within 16G3 that stabilized the reaction’s transition state and provided for substrate recognition. For a series of substrates the high product yield was due to substrate sequestration into the combining site and a low amount of product inhibition, which appears to be due to the noncongruency of the p-nitrophenyl moiety contained within the substrate and the corresponding p-nitrobenzyl group in the hapten. Furthermore, the antibody did not catalyze the racemization of the ester (structure 10) and the rate enhancement was sufficiently large to outcompete the spontaneous hydrolysis of the ester. This suggests that perhaps one of the most useful applications of abzymes will be to enhance a desired reaction that is one of a multireaction manifold. On the other hand, trying to align two catalytic residues by the bait and switch technology in order to catalyze bimolecular reactions has been less successful. The

463

phosphinate hapten (structure 14), which also contains an acid moiety, was designed to produce abzymes capable of catalyzing an aldol reaction by utilizing two positive residues in a combining site: one stabilizing the putative tetrahedral intermediate and the other stabilizing the enolate anion [36]. None of the antibodies isolated from the immunological repertoire were capable of this reaction, but one was found to catalyze the decomposition of substrate (structure 13) most likely through a retro-aldol mechanism. Heterologous immunization was proposed as a method of programming complicated combining sites [37]. To obtain antibodies capable of anilide hydrolysis, the mouse was immunized with two haptens (structures l6A and 16B): the phosphonamidate hapten (structure l6A) to sculpt the pocket for stabilization of the transition state and the pyridinium hapten (structure 16B) to introduce a basic residue for acid-base catalysis. Because none of the antibodies contained an appropriately placed nucleophile, this basic residue was incorporated into the substrate (structure 15) to give a 2 x 104 rate enhancement (pH =6.8) for the antibody reaction.

Future antibodies The question is whether the potential of has been tapped, or are there rare motifs within the immunological response that have captured. Recent investigation of X-ray crystal and computer modeling of a series of antibodies that combining sites created by similar haptens to a single motif. Five hydrolytic antibodies

antibodies generated not been structures suggests converge (including

Figure 3

# ‘PH2bW

# xGH213CW

17

A depiction of reactive immunization where the active diketone hapten 17 is trapped by a lysine residue contained within an antibody (Ab) produced during the immune response. This technique is designed to increase the subpopulation of antibodies with active site residues capable of acid-base catalysis of an aldol condensation between aldehydes and ketones (e.g. structure 18 and acetone).

464

Protein engineering

43C9 and 4867) that were raised against different phosphonate or phosphonamidate haptens contain an apolar pocket surrounded with aromatic sidechains (to bind the hapten’s aromatic ring) and charged sidechains (arginine, lysine or histidine), to help neutralize the negatively charged phosphonyl oxygens [38]. The loci of these residues provided dramatic differences in catalytic efficiency (&,43e9 = 400 min-l and &st,46G7 = 5 min-l). A convergence of motifs also occurs in monoclonal antibodies raised against a single hapten [39,40]. An entire monoclonal library was screened for catalysts of ester hydrolysis using catalytic enzyme-lined immunosorbent assay, catELISA (see below) [17]. Out of 1570 monoclonal antibodies, 970 were binders and 9 were catalytic, which illustrates the low frequency of catalysts generally found in a pool of monoclonals. The X-ray structure of three of the catalysts showed very similar binding pockets, with each having an appropriately placed tyrosine to provide stabilization of the putative tetrahedral intermediate in the combining pocket, giving rate enhancements from 103 to 105 over background. This convergence to a single motif can be advantageous if that motif is catalytically active.

One method of controlling the subpopulation of antibodies is through reactive immunization. In these experiments, the hapten contains an active group, such as a phosphonate diester [41] or 1,3-diketone [42] that, in theory, is available for covalent bond formation with antibody (Figure 3) producing cells during the immune response. For example,

Glycosidic

two catalytic antibodies were obtained in the immune repertoire raised against diketone (structure I7), and were found to contain an appropriately placed lysine in the combining site that accelerated the aldol reaction between a variety of aldehydes, for example, structure 18, and ketones via enamine formation. The rates were measured by changing the aldehyde concentration (R,,,/K, ~800 min-1 M-l) and holding the ketone’s concentration at a constant value (S%v/v), which precludes direct comparison to the background rate.

As mentioned earlier, catELISA is a modern method for screening antibody libraries for catalysts [43]. Unfortunately, the technique is still labor intensive because each antibody has to be expressed in sufficient quantities and then individually screened. A more efficient approach has been demonstrated by Janda et al. [44], who screened antibodies attached to ml3 phage particles. Antibodies were raised against hapten (Figure 4, structure 19) to catalyze glycosidic bond cleavage, where the difluoromethyl aryl glucoside substrate (structure 20) may act as a mechanism-based inhibitor. When the glycosidic bond is cut, the phenyl product is chemically active and forms a covalent bond with any nucleophile contained in the combining site (Figure 4). After removing noncatalytic antibodies, the trapped phage particle is released by disulfide bond cleavage, allowing for the recovery of the abzyme’s gene and its subsequent antibody expression. Several catalytic antibodies were obtained

bond

A method capable of screening a library of antibodies for catalysts. Antibodies were raised against hapten (structure 19) and, after subcloning into the appropriate vector, were expressed as fusion proteins displayed on ml 3 phage particles. The substrate (structure 20) is a mechanism-based inhibitor and, after activation by chemical cleavage of the glycosidic bond, it may covalently trap nucleophiles (Nu:) within the antibody pocket. The phage particle, once immobilized, allowed for the isolation and recovery of the abzyme gene.

The state

by this screening procedure and the one tested gave a greater catalytic rate enhancement for the hydrolysis of p-nitrophenyl-P-galactopyranoside compared to abzymes obtained from the traditional screening for hapten binders = 7 x 104 and Rcat/R,,,,t = 1 x 102, respectively). (&&,,,,t This method, as well as genetic selection through auxotrophic complementation [45,46], holds the greatest potential for investigating large libraries of antibody fragments for rare catalytic motifs.

Conclusions Employing the current technology to procure catalytic antibodies will yield, on average, catalysts capable of 103-105-fold rate enhancements over background, mainly as a result of sequestration of substrates into a reactive conformation or configuration within the combining site. To fulfill the promise of highly efficient, tailor-made catalysts, screening of large libraries of antibodies for catalysts will probably be required. Whether future antibodies can be designed to catalyze reactions that are mechanistically complex, as well as to bind the products weakly, remains an intriguing question because of the significant technological hurdles.

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