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Annu. Rev. Biochem. 1992. 61:29-54 Copyrighl © 1992 by Annual Reviews Inc. All righls reserved
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CATALYTIC ANTIBODIES Stephen J. Benkovic Department of Chemistry, The Pennsylvania State University, 152 Davey Laboratory, University Park, Pennsylvania 16802 KEY WORDS:
abzymes, catalytic antibodies, transition state analogs
CONTENTS INTRODUCTION . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
HISTORICAL PERSPECTi VE . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30
PERTINENT CHARACTERISTICS OF ANTIBODIES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
31
REACTION TYPES CATALYZED BY ANTIBODIES ...... . . . . . . . . ............. . . . . . ...... Antibodies As "Free Energy Traps" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . .
32 32 38 41 44 45 47 48 49 50
Bimolecular Reactions ........................................................................... Hydrolysis . . .......... . . . . . ............ .. . .............. . . . .... .......... . . . . . . . .................. . .
IMPROVING THE ODDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . Cloning ....... . .. . . . . . . ...... . . . . . ................ .. . . ....................... .... ... . . . . ..... . ......
Hapten Design...................... . . .............. . ................ . . . ................. . . . . ...... Chemical and Genetic Modification.... ................................ . . . . ............ . ...... Cofactors ........ . . . . ......... . . . . . . . . . .......... . .............................. . . ...... . ...... .....
CONCLUSION . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .
INTRODUCTION We have long been fascinated by the intricate stereospecificities and astonish ing turnover numbers of nature's catalysts, the enzymes. There exists a voluminous literature describing ingenious experiments and methodologies to delineate the structural basis of the molecular recognition inherent in enzyme substrate specificity, the origins of the enzyme's catalytic power, and the chemical mechanisms by which the substrate's transformation to product by the enzyme is achieved. With at least partial understanding of these features of enzymic catalysis has come the desire to imitate or improve, leading on one hand to the construction of macrocylic molecules of various types capable of 29
0066-4154/92/0701-0029$02.00
30
BENKOVIC
substrate discrimination and possessing at present modest catalytic power the field of supermolecular chemistry ( l-3)--and on the other hand to the modification of existing enzymes through genetic or chemical means to alter their substrate specificity without loss of catalytic efficiency-the field of
(4, 5). A third approach draws on the remarkable capacity of the immune system
protein engineering
to generate-in vast numbers-immunoglobulins that possess high affinity
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and unmatched structural specificity towards virtually any molecule (6-9) 8 (some 10 different antibody molecules are available in the primary response, a number further expanded by somatic mutation). The experimental challenge is clear: how to harness and to select, from the enormous numbers and diversity of the immunological response, molecular frameworks in the form of antibodies, inherently able not only to bind a given substrate but also to catalyze a preselected chemical transformation of it. Thus one might bypass for the time-being the thorny unsolved problems that bedevil the design of de novo catalysts: the fit of substrate to catalyst, the flexibility of the catalyst to facilitate a step-wise process, the location and strength of complementary noncovalent bonding interactions, etc. To set the stage, I begin with an abbreviated, historical perspective; a brief description of the physical and structural properties of antibodies; and a limited survey of the reaction types now known to be catalyzed by antibodies. More complete discussions can be found in other reviews
(10--15). My main intent, however, is to examine the
information available on the mechanisms of action of catalytic antibodies in order to draw parallels and contrasts to those of enzymes: How do catalytic antibodies function kinetically? For a particular chemical transformation will the same reaction mechanism be favored by both enzyme and catalytic antibody? Will catalytic antibodies use less complex kinetic sequences owing to their selection by a single immunogen? How high are their turnover numbers, and how can they be improved? These are some of the queries that owing to the rapid development of the field can now be partially answered.
HISTORICAL PERSPECTIVE The concept of inducing antibodies that would possess, in addition to their exquisite ligand specificity, catalytic potential has its roots in the seminal contributions of Pauling
(16, 17). His proposal that the ability of an enzyme to
speed up a chemical reaction stemmed from the "complementarity of the enzyme's active site structure to the activated complex" shifted the research focus from concerns about substrate-enzyme fit to a means for defining the structural requirements for binding the transition state. Since a transition state by definition has a negligible lifetime, evidence was sought for transition state stabilization in the tighter binding of inhibitors whose structures mimick-
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CATALYTIC ANTIBODIES
31
ed those of the presumed transition state relative to the weaker binding of substrate (18, 19). Many examples of such high-affinity transition-state in hibitors are now documented (20-22). At the risk of being pedantic , stabiliza tion of the transition state alone is necessary but not sufficient to give catalysis (23, 24), which requires differential binding of substrate and transition state. As we see later, the use of transition-state analogs to induce catalytic antibod ies successfully stems not only from this differential binding but other factors as well. Nevertheless, it was apparent that such inhibitors would furnish a convenient starting-point for creating catalytic antibodies (25, 26) or, even possibly by immunizing with the substrate, if the challenge was repeatedly presented to the immunological system (27). Earlier attempts (28-30), however, were thwarted by the use of polyclonal rather than monoclonal antibodies (31), and by the need for better transition-state mimics. Many earlier experiments also lacked adequate controls, for example the necessary demonstration that the immunogen acted as a competitive inhibitor of the catalysis, so that the attribution of catalytic properties to the antibody in this case is equivocal (30). PERTINENT CHARACTERISTICS OF ANTIBODIES
Antibodies are large proteins assembled in a disulfide cross-linked four-chain structure. The major serum antibody, IgG, consists of two identical heavy chains of molecular weight approximately 50,000 and two identical light chains of molecular weight 25,000 (32). Sequence comparison of monoclonal IgG proteins indicates that the carboxy-terminal half of the light chain and roughly three quarters of the heavy chain from the carboxy end show little sequence variation (33, 34). The antigen-combining site of the molecule is in the first 100 amino acids of the amino-terminal regions of both light and heavy chains, referred to as VL and VH domains , which show considerable sequence variability. Within these variable regions are short stretches of extreme amino acid sequence variation-three such regions in both the heavy and the light chains-associated with antigen recognition and designated as complementarity-determining regions. Proteolytic cleavage of the molecule on the carboxy-terminal side of the interstrand disulfide linkage connecting the light and heavy chains generates two Fab molecules, each containing an antigen-binding region. Crystallographic studies of Fab fragments, reviewed most recently by Dav ies et al (36), reveal that the immunological fold consists of two twisted stacked ,B-sheets (37), a structural motif characterizing the VB and VL do mains. One sheet has four and the other three antiparallel f3-strands related by a pseudo two-fold axis. These strands are joined at their ends by the six loops of the complementarity-determining regions, creating a key f3-barrel fold that
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32
BENKOVIC
can tolerate sequence and conformational changes in the loop region (35). On the basis of comparative studies of known antibody structures, Chothia et al have argued that there is a small repertoire of main-chain conformations "canonical structures"-for at least five of the six variable regions of antibod ies (38) whose conformation is determined by a few key residues. The area of interaction between the antigen and antibody may be relatively flat and extensive for protein antigen binding to an antibody (700-750 A2) (39-41), whereas in the case of small organic molecules the binding may occur by way of clefts whose volumes are in excess of 600 A3 (35). For small organic molecules such as fluorescein the dissociation constant of the antibody antigen complex ranges from 10-4 to 10-14 M-1 (42), which if totally coupled to drive a chemical transformation would provide a free energy change up to 20 kcal M-1, sufficient to promote most reactions in aqueous solution. The binding of antigen does not result in a global conformational change in the antibody. Rather the union is accommodated by conformational adjustments in the specific amino acid side-chains that improve the weakly binding interactions that involve hydrogen bonds, van der Waal, and electrostatic forces.
REACTION TYPES CATALYZED BY ANTIBODIES There are now ca. 50 reactions that have been catalyzed by antibodies (10). I have selected several representative examples that are instructive as to the present scope of reaction types and that also serve as my basis for discussion of mechanism. Figures 1-10 depict these reactions along with the inducing antigen to the left.
Antibodies As "Free Energy Traps" Conceptually, the reactions most susceptible to antibody catalysis would be those originally viewed as "no-mechanism" reactions because their transition states evinced little polar or radical character. Broadly speaking, these are pericyclic processes that involve bond formation through a symmetry-allowed synchronous reorganization involving the highest occupied and lowest un occupied molecular orbitals (56). These transformations include electrocyclic reactions, sigmatropic rearrangements, and cycloadditions (57), and generally are not reactions found to be c,atalyzed by enzymes. The Claisen rearrangement and Diels-Alder addition described in Figures 1 and 2 are two such examples. The former is a 3,3-sigmatropic rearrangement of chorismate to prephenate in which formation of a carbon-carbon bond is accompanied by breaking of a carbon-oxygen bond. In this case there is a biological counterpart; the same reaction is catalyzed by chorismate mutase from Escherichia coli (58). The nonenzymatic reaction has been demon-
H
· o"C-\1
.o,,�COz" O U
CO,: 0 O�O.N� o
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Figure 1
0
0
OH
0
Claisen rearrangement
(49-51).
CI CI
�CI.?
S,0 CI
-
+
Diels-Alder condensation
:«
I CI C CI CI
0
N·EI
0
Figure 2
(Top & bottom)
(52, 53).
Figure 3 (46).
Lactonization (the same antibody catalyzes lactonization and amide bond formation)
-
+
Figure 4
Amide bond formation
(47).
CH_-tNACM Annu. Rev. Biochem. 1992.61:29-54. Downloaded from www.annualreviews.org by Syracuse University Library on 04/22/13. For personal use only.
Figure 5 Transesterification (the same set of antibodies catalyze either the stereospecific hydrolysis of the R- or S- sec-phenethyl esters or the respective transesterification) (44).
Figure 6
Ester hydrolysis (43).
o o � � ¢ NOz a • m-NOz compound is a competitive inhibitor Figure 7 Amide hydrolysis (the same antibody catalyzes ester hydrolysis when the leaving alcohol is one of a series of p-substituted phenols) (45).
o
�-03 Figure 8
Decarboxylation (55).
O'
02N
CN � �O'
+
CO2
CATALYTIC ANTIBODIES
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Figure 9
f3-Elimination (48).
Figure 10
-ooc...('
35
Metallation (54).
•••
HA
O�coo-
�
HO \.. N
Figure 11 Transition state for Claisen rearrangement.
strated to occur through an asymmetric chairlike transition state in which the carbon-oxygen bond is nearly broken, whereas carbon-carbon bond formation has lagged substantially behind (59-61). On the other hand, although the E. coli enzyme-catalyzed reaction may proceed through a covalently linked intermediate based on measurements of secondary tritium (C-4 and C-5) and deuterium solvent isotope effects, the key transition state (Figure II) is also thought to be a chairlike species (62, 63). Consequently, the inducing antigen depicted in Figure 1 is a potent in hibitor of the enzyme with a K; of 0.12 fLM (64), since it closely mimics the putative bicyclic structure of the chairlike transition state shown in Figure 11. As is the case for all transition-state mimics, it is not perfect since it does not reflect either the asynchronous bond breakage and formation or the planarity of the carboxylate substituent. This mimic was used independently by two groups to produce two antibod ies that catalyzed the rearrangement of chorismate to prephenate (50, 51). Values of kcat were 2.7 min-1 and 0.072 min-1 (KMs were 260 fL M and 51
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36
BENKOVIC
JLM), illustrating the very important principle that the use of an identical immunogen does not perforce dictate isolation of an antibody with identical catalytic parameters because of the immense number of potentially catalytic antibodies. For the more reactive antibody there was no measurable deuterium solvent isotope effect, although like the mutase enzyme it catalyzed the rearrangement of the a-methyl ether, thus ruling out mechanisms involving 4-hydroxyl loss or its participation in oxirinium formation (62). The antibody catalyzed process is also stereospecific, kinetically resolving the (±) racemate of chorismate (49). There are various means of estimating the rate acceleration achieved by restricting the conformational freedom of substrate molecules. Since the results are superficially discordant, one should view them as merely establish ing a range for reference. One method employs quantum mechanical calcula tions of the entropy loss (presuming Ml* is fixed) to determine the effect on rate, i.e. the rate constant is proportional to flS+/R. For a reaction model f�aturing the conversion of succinic acid to its anhydride, an entropy loss of ca. 15 eu results from the disappearance of two degrees of internal rotation and a low-entropy asymmetrical and symmetrical bending mode (65). This translates to a rate advantage of ca. 40 [7.5 entropy units (eu)] per rotation. A similar calculation based on cyclopentadiene dimerization provides a value of ca. 10 as the rate advantage for freezing out of a single bond rotation (66). On the other hand, experimental observations that compare various in tramolecular cyclizations furnish estimates of 102-104-fold rate enhancements (67, 68) associated with a single frozen rotation in the absence of steric strain or electronic effects. The discrepancy between theory and experiment is not as grave as it might first appear; the inclusion of the loss-of-bending motions in the calculation provides entropy losses up to 14 eu, or a rate factor of 103 for a single rotation (69). Consequently, the restriction of chorismate by either antibody into a more reactive pseudo diaxial chair conformation with appro priate alignment of reacting bonds (a point we return to later) alone may be sufficient to rationalize the observed rate accelerations of up to 104 over background, a factor only ca. 102-fold less than the enzyme's. The Diels-Alder reaction provides a second example of a transformation proceeding through a highly ordered, entropy disfavored, transition state with little charge development (70, 71), which must more closely resemble the product than the conjugated diene and olefin starting materials. However, inducing antibodies to the product itself would produce binding sites subject to severe product inhibition. Two strategies have been created to circumvent this undesirable property: (a) catalyze the formation of an initial, unstable bicyclic intermediate that subsequently rearranges or, as in Figure 2 (top) extrudes S02, to yield the ultimate product; or (b) incorporate into the transition-state analog a molecular constraint that restricts the analog to a
37
CATALYTIC ANTIBODIES
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Figure J 2 reaction.
Transition state for Diels-Alder
higher-energy conformational state than the product. In Figure 2 (bottom) the ethano bridge locks the cyclohexene ring of the hapten into a conformation that resembles the pericyclic transition state for the Diels-Alder reaction (Figure 12) but that corresponds to a less favored boat conformation of the product. Both strategies led to antibodies that promoted multiple turnovers. It is worth noting that the antibodies used to catalyze the reaction of tetrachlor othiophene dioxide with N-ethylmaleimide had to first be protected from the reactivity of the former reagent with lysine by reductive methylation, a protocol that may be necessary with other substrates capable of protein modification. From analyses and experimental comparisons along the lines discussed earlier , various estimates have been projected for the advantage of converting a solution bimolecular process to an intramolecular one through formation of a catalyst bisubstrate complex. If we use the same quantum mechanical calculation, the outcome depends on whether the complexed state is loose internal and rotational degrees of freedom of the complex offsetting the loss of translational entropy--or tight-internal degrees of freedom converted to bending motions. For a loose complex the entropy loss is only 4.1 eu, corresponding to ca. 8-fold rate acceleration (65), similar to a value reached earlier by analysis of the combination of two bromine atoms (72, 73). For a tight complex the loss of entropy is an additional 35 eu, equivalent to an overall rate acceleration of 108 M, similar to that obtained from the analysis of the cyclopentadiene dimerization (66). (The standard state is 1 M in reactants, 25°C.)
These calculations presume that the orientation of the reacting groups is favorable for reaction. There is little theoretical or experimental support for the hypothesis that the orientation of the reactive groups need be confined to only a very narrow spatial range (73); in fact theoretically there is little entropy to be gained by restriction of vibrational amplitudes (65, 74) and experimentally the orientation of reactive moieties can deviate by angular displacements of > 10° with no effect on reaction rates (75). (Of course, gross misalignment of reactive moieties-pointing in opposite directions-would not be accommodated.) From experimental comparisons of intramolecular and intermolecular reac tions operating by the same mechanism, an effective molarity parameter has ,
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38
BENKOVIC
been devised to quantify intramolecularity. The ratio kintralkinter where k represents the first- and second-order rate constant for the respective pro cesses ranges from < 1 M to > 1010 M, reflecting ring size, reaction type, etc (76). For our purposes, a value of 108 M represents an upper limit for a tight, strain-free complex. The antibodies that catalyze the Diels-Alder reactions exhibit effective molarities of> 110 M (Figure 2, top) and 0.35 M or 335 M relative to buffer or acetonitrile solvent (Figure 2, bottom). For both antibodies the binding of the final product molecule was 100--1000-fold less than the hapten molecule, further confirming the validity of the design strategy. It will be of consider able interest to determine the mechanistic characteristics of these antibody catalyzed transformations, since there is no known enzyme counterpart. The intramolecular cyclization reaction (Figure 3) presents an additional opportunity, namely for general acid-base catalysis, as well as a need to reduce the rotational entropy of the substrate in order to maximize the rate of lactonization. The ring closure in the absence of antibody is specific-base catalyzed, consistent with nucleophilic attack by alkoxide ion generated from the hydroxy ester. The pH-rate profiles for kcat and kca,lKM for the antibody catalyzed process likewise are first-order in hydroxide ion (pH 7-10), provid ing no evidence for the dissociation of a participating binding-site residue. The rate acceleration is 790, suggesting that values of 102_104 may be typical for uncatalyzed intramolecular rearrangements within the antibody-binding pocket. Most importantly, this antibody catalyzed the enantioselective cyclization of one stereoisomer of the racemic hydroxy ester with >94% enantiomeric excess as determined by I H NMR in the presence of chiral shift reagents. This early example demonstrated the feasibility of catalytic antibod ies for chemical transformations that require precise stereochemical control. Two examples that fit into this class of reactions should be mentioned in passing, namely the photochemically allowed dimerization of substituted olefins (77) and the photo-induced cleavage of thymidine dimers (78). Both take advantage of the ubiquitous decoration of antibody-binding sites with tryptophans and use the indole side-chain as a photo sensitizer. In the case of the cleavage process, the rate rivaled that of the E. coli DNA photolyase (kcat of 1.2 min-I, antibody vs kcat of 3.4 min-I, enzyme). More examples of catalytic antibody-induced excited state chemistry should be pursued.
Bimolecular Reactions Two general classifications of kinetic sequences, distinguishable by steady state kinetics, describe bimolecular enzyme-catalyzed reactions: sequential and ping-pong. For the sequential process, the chemistry of bond formation and cleavage takes place within the bisubstrate enzyme ternary complex; in contrast, for the ping-pong sequence, more molecular finesse is required, with
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CATALYTIC ANTIBODIES
39
the enzyme acting to shuttle through covalent attachment an isolated fragment of one substrate for chemical union with the second. Of course within the sequential pathways there are variations imposed by whether the binding of either substrate is independent of the other, is at kinetic equilibrium relative to turnover, etc. Two bimolecular antibody-catalyzed reactions now have been examined in sufficient detail to permit their unequivocal classification. The initial velocity of the enantioselective reaction of l,4-phenylenedia mine with the lactone shown in Figure 4 was studied over a range of substrate concentrations for both the lactone and l,4-phenylenediamine substrates. The resulting Lineweaver-Burke plots showed an intersection pattern consistent with a random equilibrium process. Since the value of Kj for the phosphonate hapten was also accurately measured from its competitive inhibition of the cyclization reaction along with the respective KMs of lactone and p phenylenediamine, one could estimate the extent to which the factor of 108 M noted above might be captured in the antibody reaction from a reaction cycle (Figure 13) based on transition-state theory (79). The values of KA, KL, and KT correspond to the KM (1,4-phenylenedia mine), KM (lactone), and Kj (phosphonate hapten) in so far as the immunogen resembles the transition state for the reaction. The more favorable binding of the phosphonate (75 nM) relative to the two substrates (1.2 mM for the diamine and 4. 9 mM for the lactone) provides an advantage of 155 M for the bimolecular reaction and 1000 for the previously discussed cyclization reac tion. The observed ratio of katJkN (equivalent to kcatlkuncat) is 16 M and 790, in fair agreement with the theoretical estimates, which ignore the inadequacies of the transition-state mimic. Whereas the weaker-than-expected binding of transition-state analogs to enzymes has been attributed to either unfavor able steric interactions (a form of destabilization) or failure to form bind ing-site contacts (a form of unproductive binding) for antibodies, the transi tion-state analog acts as both model and mold. Thus the question of
Ab+L+A
1�
* KN
-
KLKA
Ab'L'A
* KAb
-
[LA1*+Ab
1�
-
P+Ab
KT
[AbLA1*
-
P+Ab
Figure 13 Reaction cycle to calculate the rate acceleration of the antibody-catalyzed reaction from the relative affinity of the antibody for the transition state versus the reactant (47).
40
BENKOVIC
fit is much less important than the accurate mimicry of the stereochemical and electronic characteristics of the transition state for the reaction in question. A compilation (86) of 18 KMIKi ratios, where KM is the Michaelis constant for substrate dissociation and Ki is the dissociation constant for the phosphon ate inhibitor for predominantly antibody-catalyzed ester hydrolysis (80-85), showed a linear though scattered correlation (slope 0.86, R2 0.80) with the ratio of kaJkN. The values of kab1kN range from 30 to 106 M and KMIKi from 4 to 3 X 104 M, (H20 at I M), with the former values roughly within one or two orders of magnitude of the latter (86). Since it is unlikely that the ratio of KMIKi will exceed 109 M (although this is more than respectable) , it is clear that rate-enhancements above this limit probably arise from the active participation of amino acid side-chains or added cofactors within the anti body-binding site. Positive deviations from this relationship, as we shall see, indeed are suggestive of more complex mechanistic processes. The transesterification reaction (Figure 5) is formally a bimolecular sub stitution reaction that like the aforementioned aminolysis reaction must com pete with the hydrolysis of the ester. Antibodies that previously had been induced with racemic phosphonate to catalyze the hydrolysis of the esters of R- and S- sec-phenethyl alcohols fell into two classes: a group that acted on the R- and a second set that acted on the S-substrates. One antibody, de signated 21H3, which was an S-specific esterase, also was found to catalyze the transesterification reaction in a mixture of water and 10% dimethyl sulfoxide through the two-step process shown in Figure 14. Remarkably, this antibody uses a ping-pong kinetic sequence, which is not limited to the vinyl ester but includes substituted benzylic esters as well. Compelling evidence adduced for the ping-pong s�quence includes: (a) dou ble reciprocal initial velocity plots that are a family of parallel lines; (b) inhibition patterns in which the ester product is a noncompetitive inhibitor of the alcohol substrate or the first alcohol product released competitively in hibits addition of the second alcohol; (c) the rapid, stoichiometric reaction of 21H3 with the p-nitrophenylester; and (d) the labeling of the antibody with a 14C radiolabel located in the acyl portion of the ester.
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=
o
21H3 +
21H3
)l + R
=
CH3CHO
0 H, CH3 o HO .H H3+ JlO R 21H3 )lR+ � CH3 � 21
V
whereR�
H CH3 N 'l( 'Y.". o
Figure 14
� V
�
Ping-pong kinetic sequence for transesterification reaction.
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CATALYTIC ANTmODIES
41
A measurement of the reaction rate in the absence of antibody was com plicated by hydrolytic cleavage of the ester at pHs at which sufficient alkoxide ion could be generated for observable transesterification. The comparison of kN and kab is further compromised by the fact that the mechanisms in the two cases are different, but with these caveats, a minimal estimate of lOs-106 M was obtained from the antibody-catalyzed rate for transesterification of the vinyl ester with sec-phenethyl alcohol. A common feature ascribed to protein conformation flexibility and often associated with enzymic catalysis is the induced fit of substrate to stabilize a rare, reactive conformer. The presence of sec-phenethyl alcohol provides a route for transesterification but also increases the esterase activity 1O-fold (J. Stewart, unpublished results). Similarly, surrogates devoid of an accepting hydroxyl, e.g. sec-phenethyl chloride and bromide, increase the rate of hydrolysis (VIK) of the vinyl ester by the antibody by a factor of 10. The simple induced-fit interpretation (88) is that in the absence of the surrogate, the antibody has either a conformation in which bound ester or the acyl intermediate is less accessible to water, or key transition states in which the acylationldeacylation pathway is less stabilized. The induction of an antibody capable of a ping-pong kinetic sequence would not at first glance appear to be implicit in the hapten design. But recall that these antibodies were produced in response to a monoesterified phosphonic acid derivative (Figure 6) with the intent to elicit esterase activity. If transesterification activity were the initial target, then the hapten should have been a diesterified phosphonate with either similar or dissimilar alcohol moieties. This would provide sufficient space in a programmed binding site for the simultaneous binding of both ester and alcohol to the antibody. One might argue, then, that in order for the transesterification reaction to be catalyzed by the present set of antibodies, it must follow a ping-pong se quence since the acceptor alcohol must occupy the same volume in space as the alcohol portion of the ester. What is remarkable then is the induction of a nucleophile at the binding site of the antibody in proper juxtaposition, with an appropriate pKa, and with a balanced acylation/deacylation reactivity to catalyze the transesterification reaction at such high efficiency.
Hydrolysis The antibody, 43C9, which catalyzes the hydrolysis of the p-nitroanilide (Figure 7) and a series of related aromatic esters (pNOz, pCI, pCH3, pCH3CO), has been the subject of an extensive number of experiments to establish its mechanism of action (89). The steady-state Michaelis-Menten parameters, kcatlKM and kcat> as a function of pH for both the p-nitroanilide and ester substrates, are exhibited in Figure 15. At high pH the observed kcat exceeds the ratio of K/KM by > lOO-fold. The data can be fit either to a reaction mechanism involving the titration of a group whose dissociation (pKa
42
BENKOVIC
1 00
§ :;:,
�
---
IV 0 ..\0: ... 0
iii
0 ..\0: .... 0
iii
0 ..\0:
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til
..9
iii
2
0 ..10:
1
..9
til
-2 -3
0 -1
6
7
9
8
10
11
-4 7
8
9
10
11
pH
pH
Figure 15 pH-rate profiles for the hydrolysis of the p-nitrophenylester (left panel) and p nitroanilide (right panel) by antibody, 43C9 (see Figure 7) (89). =
9.0) promotes substrate hydrolysis, or to a mechanism that features a
change in the rate-limiting step around an intermediate antibody-bound spe cies due to changes in pH. Support for the latter sequence stems from several lines of evidence. Firstly, that a true pKa is not being observed in the pH-rate profiles is in accord with the observations that >
(a) the plateau rate encountered in kcat at pH 9.0 for hydrolysis of the p-nitroester is a consequence of the slow dissocia
tion of the p-nitrophenol product; (b) the rate constants for binding of p nitrophenol as well as the dissociation constant Ki (for the p-nitroanilide as a competitive inhibitor of ester hydrolysis) are pH independent over the range of measurement ;
(c) slow dissociation of p-nitrophenol should perturb the pKa of a group in common to both profiles by ca. 0.7 pH units for the ester relative to the anilide (90). Secondly, measurement of the pH-kcat and kca/KM profiles for both the hydrolysis of the anilide and the p-CI ester in deuterium oxide revealed no deuterium solvent isotope effect on kcat or kca/KM at pH > 9.0 but a substantial effect (k2H20/k2D20 = 2-4) on kcat and kca/KM at pH < 9.0 (91, 92). These observations rule out a simple general base-catalyzed hydrolytic mechanism involving a single dissociable group, and are only in accord with two different rate processes defining the apparent pKa. Evidence for the presence of an acyl intermediate sought by both stopped flow and rapid quench methods did not reveal any accumulation above
(89, 92). However, the hydrolysis of a series 43C9 gave kcat values above pH 9.0 , which were
undetectable steady-state levels of aromatic esters by
strongly dependent on the electronic nature of the p-substituent. The Hammett p value is
2.3, consistent with values of 2-3 observed for attack on acyl esters
CATALYTIC ANTIBODIES
Figure 16
43
Kinetic reaction sequence for es
ter and anilide hydrolysis catalyzed by 43C9
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(89).
by nitrogen nucleophiles (93) but not values of 1.0-1. 2 and 0.5-0.7 found for nucleophilic attack by oxyanions or general base catalysis (94). This observa tion, coupled with a lack of 180 exchange from H2 180 into unreacted anilide in the presence of antibody, also rules out the accumulation of a symmetrical tetrahedral intermediate. The weight of the data then favor the reaction sequence given in Figure 16, where S is either anilide or ester substrate, I is the acylated antibody plus P2, PI is the acid product, and P2 the phenol or aniline. The pH-rate profiles arise from a change in rate-limiting step, from product release (ester, kcat) or acylation (anilide, kcatlKM) at high pH, to deacylation (kcat and kcatlKM) at low pH. From the primary sequence of the antibody, 43C9, the most likely candidate for the nucleophile is a histidine located in the L3 loop of the light chain. However, the appearance of a large deuterium solvent isotope effect on the deacylation leg is not in accord with attack by hydroxide on an acyl intermediate, but suggests that a second basic group on the antibody may be acting to catalyze deacylation. Its pKa, like that of the nucleophile, is outside the range of the pH-rate profiles. The relative importance of the various kinetic steps in antibody turnover can be more easily visualized through the aid of a free energy (LlC*) reaction coordinate diagram (Figure 17). There are several striking features in the two profiles. The first is the general uneveness in the LlC* barriers for the various internal antibody-substrate complexes and their respective transition states. A more optimal situation would balance the differences in LlC:): between the external and internal ground states as well as their respective transition states-as is the case for enzymes operating at optimal flux-so that no single AC+ barrier would be egregious (95). A second is the high stability of the Ab·PI·P2 product complex relative to the respective uncomplexed substrates. It is this tight product complex that ultimately limits the rate of ester hydroly sis (kcat) and also is responsible for the lack of Ab·I accumulation. The distortion introduced by tight product binding has been remedied by chang ing the nature of the p-substituent, e.g. pN02 to pCl, but with a drop in the acylation rate (92). A third feature is the increased kinetic
44
BENKOVIC
15 --. '. l
10 .....
"0 E
! \ :"r: ;\
i 1
5
I f f I I I I I .:�.
-....
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m u
� ......
0
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Further
Quick links to online content
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CATALYTIC ANTIBODIES Stephen J. Benkovic Department of Chemistry, The Pennsylvania State University, 152 Davey Laboratory, University Park, Pennsylvania 16802 KEY WORDS:
abzymes, catalytic antibodies, transition state analogs
CONTENTS INTRODUCTION . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
HISTORICAL PERSPECTi VE . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30
PERTINENT CHARACTERISTICS OF ANTIBODIES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
31
REACTION TYPES CATALYZED BY ANTIBODIES ...... . . . . . . . . ............. . . . . . ...... Antibodies As "Free Energy Traps" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . .
32 32 38 41 44 45 47 48 49 50
Bimolecular Reactions ........................................................................... Hydrolysis . . .......... . . . . . ............ .. . .............. . . . .... .......... . . . . . . . .................. . .
IMPROVING THE ODDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . Cloning ....... . .. . . . . . . ...... . . . . . ................ .. . . ....................... .... ... . . . . ..... . ......
Hapten Design...................... . . .............. . ................ . . . ................. . . . . ...... Chemical and Genetic Modification.... ................................ . . . . ............ . ...... Cofactors ........ . . . . ......... . . . . . . . . . .......... . .............................. . . ...... . ...... .....
CONCLUSION . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .
INTRODUCTION We have long been fascinated by the intricate stereospecificities and astonish ing turnover numbers of nature's catalysts, the enzymes. There exists a voluminous literature describing ingenious experiments and methodologies to delineate the structural basis of the molecular recognition inherent in enzyme substrate specificity, the origins of the enzyme's catalytic power, and the chemical mechanisms by which the substrate's transformation to product by the enzyme is achieved. With at least partial understanding of these features of enzymic catalysis has come the desire to imitate or improve, leading on one hand to the construction of macrocylic molecules of various types capable of 29
0066-4154/92/0701-0029$02.00
30
BENKOVIC
substrate discrimination and possessing at present modest catalytic power the field of supermolecular chemistry ( l-3)--and on the other hand to the modification of existing enzymes through genetic or chemical means to alter their substrate specificity without loss of catalytic efficiency-the field of
(4, 5). A third approach draws on the remarkable capacity of the immune system
protein engineering
to generate-in vast numbers-immunoglobulins that possess high affinity
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and unmatched structural specificity towards virtually any molecule (6-9) 8 (some 10 different antibody molecules are available in the primary response, a number further expanded by somatic mutation). The experimental challenge is clear: how to harness and to select, from the enormous numbers and diversity of the immunological response, molecular frameworks in the form of antibodies, inherently able not only to bind a given substrate but also to catalyze a preselected chemical transformation of it. Thus one might bypass for the time-being the thorny unsolved problems that bedevil the design of de novo catalysts: the fit of substrate to catalyst, the flexibility of the catalyst to facilitate a step-wise process, the location and strength of complementary noncovalent bonding interactions, etc. To set the stage, I begin with an abbreviated, historical perspective; a brief description of the physical and structural properties of antibodies; and a limited survey of the reaction types now known to be catalyzed by antibodies. More complete discussions can be found in other reviews
(10--15). My main intent, however, is to examine the
information available on the mechanisms of action of catalytic antibodies in order to draw parallels and contrasts to those of enzymes: How do catalytic antibodies function kinetically? For a particular chemical transformation will the same reaction mechanism be favored by both enzyme and catalytic antibody? Will catalytic antibodies use less complex kinetic sequences owing to their selection by a single immunogen? How high are their turnover numbers, and how can they be improved? These are some of the queries that owing to the rapid development of the field can now be partially answered.
HISTORICAL PERSPECTIVE The concept of inducing antibodies that would possess, in addition to their exquisite ligand specificity, catalytic potential has its roots in the seminal contributions of Pauling
(16, 17). His proposal that the ability of an enzyme to
speed up a chemical reaction stemmed from the "complementarity of the enzyme's active site structure to the activated complex" shifted the research focus from concerns about substrate-enzyme fit to a means for defining the structural requirements for binding the transition state. Since a transition state by definition has a negligible lifetime, evidence was sought for transition state stabilization in the tighter binding of inhibitors whose structures mimick-
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CATALYTIC ANTIBODIES
31
ed those of the presumed transition state relative to the weaker binding of substrate (18, 19). Many examples of such high-affinity transition-state in hibitors are now documented (20-22). At the risk of being pedantic , stabiliza tion of the transition state alone is necessary but not sufficient to give catalysis (23, 24), which requires differential binding of substrate and transition state. As we see later, the use of transition-state analogs to induce catalytic antibod ies successfully stems not only from this differential binding but other factors as well. Nevertheless, it was apparent that such inhibitors would furnish a convenient starting-point for creating catalytic antibodies (25, 26) or, even possibly by immunizing with the substrate, if the challenge was repeatedly presented to the immunological system (27). Earlier attempts (28-30), however, were thwarted by the use of polyclonal rather than monoclonal antibodies (31), and by the need for better transition-state mimics. Many earlier experiments also lacked adequate controls, for example the necessary demonstration that the immunogen acted as a competitive inhibitor of the catalysis, so that the attribution of catalytic properties to the antibody in this case is equivocal (30). PERTINENT CHARACTERISTICS OF ANTIBODIES
Antibodies are large proteins assembled in a disulfide cross-linked four-chain structure. The major serum antibody, IgG, consists of two identical heavy chains of molecular weight approximately 50,000 and two identical light chains of molecular weight 25,000 (32). Sequence comparison of monoclonal IgG proteins indicates that the carboxy-terminal half of the light chain and roughly three quarters of the heavy chain from the carboxy end show little sequence variation (33, 34). The antigen-combining site of the molecule is in the first 100 amino acids of the amino-terminal regions of both light and heavy chains, referred to as VL and VH domains , which show considerable sequence variability. Within these variable regions are short stretches of extreme amino acid sequence variation-three such regions in both the heavy and the light chains-associated with antigen recognition and designated as complementarity-determining regions. Proteolytic cleavage of the molecule on the carboxy-terminal side of the interstrand disulfide linkage connecting the light and heavy chains generates two Fab molecules, each containing an antigen-binding region. Crystallographic studies of Fab fragments, reviewed most recently by Dav ies et al (36), reveal that the immunological fold consists of two twisted stacked ,B-sheets (37), a structural motif characterizing the VB and VL do mains. One sheet has four and the other three antiparallel f3-strands related by a pseudo two-fold axis. These strands are joined at their ends by the six loops of the complementarity-determining regions, creating a key f3-barrel fold that
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32
BENKOVIC
can tolerate sequence and conformational changes in the loop region (35). On the basis of comparative studies of known antibody structures, Chothia et al have argued that there is a small repertoire of main-chain conformations "canonical structures"-for at least five of the six variable regions of antibod ies (38) whose conformation is determined by a few key residues. The area of interaction between the antigen and antibody may be relatively flat and extensive for protein antigen binding to an antibody (700-750 A2) (39-41), whereas in the case of small organic molecules the binding may occur by way of clefts whose volumes are in excess of 600 A3 (35). For small organic molecules such as fluorescein the dissociation constant of the antibody antigen complex ranges from 10-4 to 10-14 M-1 (42), which if totally coupled to drive a chemical transformation would provide a free energy change up to 20 kcal M-1, sufficient to promote most reactions in aqueous solution. The binding of antigen does not result in a global conformational change in the antibody. Rather the union is accommodated by conformational adjustments in the specific amino acid side-chains that improve the weakly binding interactions that involve hydrogen bonds, van der Waal, and electrostatic forces.
REACTION TYPES CATALYZED BY ANTIBODIES There are now ca. 50 reactions that have been catalyzed by antibodies (10). I have selected several representative examples that are instructive as to the present scope of reaction types and that also serve as my basis for discussion of mechanism. Figures 1-10 depict these reactions along with the inducing antigen to the left.
Antibodies As "Free Energy Traps" Conceptually, the reactions most susceptible to antibody catalysis would be those originally viewed as "no-mechanism" reactions because their transition states evinced little polar or radical character. Broadly speaking, these are pericyclic processes that involve bond formation through a symmetry-allowed synchronous reorganization involving the highest occupied and lowest un occupied molecular orbitals (56). These transformations include electrocyclic reactions, sigmatropic rearrangements, and cycloadditions (57), and generally are not reactions found to be c,atalyzed by enzymes. The Claisen rearrangement and Diels-Alder addition described in Figures 1 and 2 are two such examples. The former is a 3,3-sigmatropic rearrangement of chorismate to prephenate in which formation of a carbon-carbon bond is accompanied by breaking of a carbon-oxygen bond. In this case there is a biological counterpart; the same reaction is catalyzed by chorismate mutase from Escherichia coli (58). The nonenzymatic reaction has been demon-
H
· o"C-\1
.o,,�COz" O U
CO,: 0 O�O.N� o
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Figure 1
0
0
OH
0
Claisen rearrangement
(49-51).
CI CI
�CI.?
S,0 CI
-
+
Diels-Alder condensation
:«
I CI C CI CI
0
N·EI
0
Figure 2
(Top & bottom)
(52, 53).
Figure 3 (46).
Lactonization (the same antibody catalyzes lactonization and amide bond formation)
-
+
Figure 4
Amide bond formation
(47).
CH_-tNACM Annu. Rev. Biochem. 1992.61:29-54. Downloaded from www.annualreviews.org by Syracuse University Library on 04/22/13. For personal use only.
Figure 5 Transesterification (the same set of antibodies catalyze either the stereospecific hydrolysis of the R- or S- sec-phenethyl esters or the respective transesterification) (44).
Figure 6
Ester hydrolysis (43).
o o � � ¢ NOz a • m-NOz compound is a competitive inhibitor Figure 7 Amide hydrolysis (the same antibody catalyzes ester hydrolysis when the leaving alcohol is one of a series of p-substituted phenols) (45).
o
�-03 Figure 8
Decarboxylation (55).
O'
02N
CN � �O'
+
CO2
CATALYTIC ANTIBODIES
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Figure 9
f3-Elimination (48).
Figure 10
-ooc...('
35
Metallation (54).
•••
HA
O�coo-
�
HO \.. N
Figure 11 Transition state for Claisen rearrangement.
strated to occur through an asymmetric chairlike transition state in which the carbon-oxygen bond is nearly broken, whereas carbon-carbon bond formation has lagged substantially behind (59-61). On the other hand, although the E. coli enzyme-catalyzed reaction may proceed through a covalently linked intermediate based on measurements of secondary tritium (C-4 and C-5) and deuterium solvent isotope effects, the key transition state (Figure II) is also thought to be a chairlike species (62, 63). Consequently, the inducing antigen depicted in Figure 1 is a potent in hibitor of the enzyme with a K; of 0.12 fLM (64), since it closely mimics the putative bicyclic structure of the chairlike transition state shown in Figure 11. As is the case for all transition-state mimics, it is not perfect since it does not reflect either the asynchronous bond breakage and formation or the planarity of the carboxylate substituent. This mimic was used independently by two groups to produce two antibod ies that catalyzed the rearrangement of chorismate to prephenate (50, 51). Values of kcat were 2.7 min-1 and 0.072 min-1 (KMs were 260 fL M and 51
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36
BENKOVIC
JLM), illustrating the very important principle that the use of an identical immunogen does not perforce dictate isolation of an antibody with identical catalytic parameters because of the immense number of potentially catalytic antibodies. For the more reactive antibody there was no measurable deuterium solvent isotope effect, although like the mutase enzyme it catalyzed the rearrangement of the a-methyl ether, thus ruling out mechanisms involving 4-hydroxyl loss or its participation in oxirinium formation (62). The antibody catalyzed process is also stereospecific, kinetically resolving the (±) racemate of chorismate (49). There are various means of estimating the rate acceleration achieved by restricting the conformational freedom of substrate molecules. Since the results are superficially discordant, one should view them as merely establish ing a range for reference. One method employs quantum mechanical calcula tions of the entropy loss (presuming Ml* is fixed) to determine the effect on rate, i.e. the rate constant is proportional to flS+/R. For a reaction model f�aturing the conversion of succinic acid to its anhydride, an entropy loss of ca. 15 eu results from the disappearance of two degrees of internal rotation and a low-entropy asymmetrical and symmetrical bending mode (65). This translates to a rate advantage of ca. 40 [7.5 entropy units (eu)] per rotation. A similar calculation based on cyclopentadiene dimerization provides a value of ca. 10 as the rate advantage for freezing out of a single bond rotation (66). On the other hand, experimental observations that compare various in tramolecular cyclizations furnish estimates of 102-104-fold rate enhancements (67, 68) associated with a single frozen rotation in the absence of steric strain or electronic effects. The discrepancy between theory and experiment is not as grave as it might first appear; the inclusion of the loss-of-bending motions in the calculation provides entropy losses up to 14 eu, or a rate factor of 103 for a single rotation (69). Consequently, the restriction of chorismate by either antibody into a more reactive pseudo diaxial chair conformation with appro priate alignment of reacting bonds (a point we return to later) alone may be sufficient to rationalize the observed rate accelerations of up to 104 over background, a factor only ca. 102-fold less than the enzyme's. The Diels-Alder reaction provides a second example of a transformation proceeding through a highly ordered, entropy disfavored, transition state with little charge development (70, 71), which must more closely resemble the product than the conjugated diene and olefin starting materials. However, inducing antibodies to the product itself would produce binding sites subject to severe product inhibition. Two strategies have been created to circumvent this undesirable property: (a) catalyze the formation of an initial, unstable bicyclic intermediate that subsequently rearranges or, as in Figure 2 (top) extrudes S02, to yield the ultimate product; or (b) incorporate into the transition-state analog a molecular constraint that restricts the analog to a
37
CATALYTIC ANTIBODIES
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Figure J 2 reaction.
Transition state for Diels-Alder
higher-energy conformational state than the product. In Figure 2 (bottom) the ethano bridge locks the cyclohexene ring of the hapten into a conformation that resembles the pericyclic transition state for the Diels-Alder reaction (Figure 12) but that corresponds to a less favored boat conformation of the product. Both strategies led to antibodies that promoted multiple turnovers. It is worth noting that the antibodies used to catalyze the reaction of tetrachlor othiophene dioxide with N-ethylmaleimide had to first be protected from the reactivity of the former reagent with lysine by reductive methylation, a protocol that may be necessary with other substrates capable of protein modification. From analyses and experimental comparisons along the lines discussed earlier , various estimates have been projected for the advantage of converting a solution bimolecular process to an intramolecular one through formation of a catalyst bisubstrate complex. If we use the same quantum mechanical calculation, the outcome depends on whether the complexed state is loose internal and rotational degrees of freedom of the complex offsetting the loss of translational entropy--or tight-internal degrees of freedom converted to bending motions. For a loose complex the entropy loss is only 4.1 eu, corresponding to ca. 8-fold rate acceleration (65), similar to a value reached earlier by analysis of the combination of two bromine atoms (72, 73). For a tight complex the loss of entropy is an additional 35 eu, equivalent to an overall rate acceleration of 108 M, similar to that obtained from the analysis of the cyclopentadiene dimerization (66). (The standard state is 1 M in reactants, 25°C.)
These calculations presume that the orientation of the reacting groups is favorable for reaction. There is little theoretical or experimental support for the hypothesis that the orientation of the reactive groups need be confined to only a very narrow spatial range (73); in fact theoretically there is little entropy to be gained by restriction of vibrational amplitudes (65, 74) and experimentally the orientation of reactive moieties can deviate by angular displacements of > 10° with no effect on reaction rates (75). (Of course, gross misalignment of reactive moieties-pointing in opposite directions-would not be accommodated.) From experimental comparisons of intramolecular and intermolecular reac tions operating by the same mechanism, an effective molarity parameter has ,
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38
BENKOVIC
been devised to quantify intramolecularity. The ratio kintralkinter where k represents the first- and second-order rate constant for the respective pro cesses ranges from < 1 M to > 1010 M, reflecting ring size, reaction type, etc (76). For our purposes, a value of 108 M represents an upper limit for a tight, strain-free complex. The antibodies that catalyze the Diels-Alder reactions exhibit effective molarities of> 110 M (Figure 2, top) and 0.35 M or 335 M relative to buffer or acetonitrile solvent (Figure 2, bottom). For both antibodies the binding of the final product molecule was 100--1000-fold less than the hapten molecule, further confirming the validity of the design strategy. It will be of consider able interest to determine the mechanistic characteristics of these antibody catalyzed transformations, since there is no known enzyme counterpart. The intramolecular cyclization reaction (Figure 3) presents an additional opportunity, namely for general acid-base catalysis, as well as a need to reduce the rotational entropy of the substrate in order to maximize the rate of lactonization. The ring closure in the absence of antibody is specific-base catalyzed, consistent with nucleophilic attack by alkoxide ion generated from the hydroxy ester. The pH-rate profiles for kcat and kca,lKM for the antibody catalyzed process likewise are first-order in hydroxide ion (pH 7-10), provid ing no evidence for the dissociation of a participating binding-site residue. The rate acceleration is 790, suggesting that values of 102_104 may be typical for uncatalyzed intramolecular rearrangements within the antibody-binding pocket. Most importantly, this antibody catalyzed the enantioselective cyclization of one stereoisomer of the racemic hydroxy ester with >94% enantiomeric excess as determined by I H NMR in the presence of chiral shift reagents. This early example demonstrated the feasibility of catalytic antibod ies for chemical transformations that require precise stereochemical control. Two examples that fit into this class of reactions should be mentioned in passing, namely the photochemically allowed dimerization of substituted olefins (77) and the photo-induced cleavage of thymidine dimers (78). Both take advantage of the ubiquitous decoration of antibody-binding sites with tryptophans and use the indole side-chain as a photo sensitizer. In the case of the cleavage process, the rate rivaled that of the E. coli DNA photolyase (kcat of 1.2 min-I, antibody vs kcat of 3.4 min-I, enzyme). More examples of catalytic antibody-induced excited state chemistry should be pursued.
Bimolecular Reactions Two general classifications of kinetic sequences, distinguishable by steady state kinetics, describe bimolecular enzyme-catalyzed reactions: sequential and ping-pong. For the sequential process, the chemistry of bond formation and cleavage takes place within the bisubstrate enzyme ternary complex; in contrast, for the ping-pong sequence, more molecular finesse is required, with
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CATALYTIC ANTIBODIES
39
the enzyme acting to shuttle through covalent attachment an isolated fragment of one substrate for chemical union with the second. Of course within the sequential pathways there are variations imposed by whether the binding of either substrate is independent of the other, is at kinetic equilibrium relative to turnover, etc. Two bimolecular antibody-catalyzed reactions now have been examined in sufficient detail to permit their unequivocal classification. The initial velocity of the enantioselective reaction of l,4-phenylenedia mine with the lactone shown in Figure 4 was studied over a range of substrate concentrations for both the lactone and l,4-phenylenediamine substrates. The resulting Lineweaver-Burke plots showed an intersection pattern consistent with a random equilibrium process. Since the value of Kj for the phosphonate hapten was also accurately measured from its competitive inhibition of the cyclization reaction along with the respective KMs of lactone and p phenylenediamine, one could estimate the extent to which the factor of 108 M noted above might be captured in the antibody reaction from a reaction cycle (Figure 13) based on transition-state theory (79). The values of KA, KL, and KT correspond to the KM (1,4-phenylenedia mine), KM (lactone), and Kj (phosphonate hapten) in so far as the immunogen resembles the transition state for the reaction. The more favorable binding of the phosphonate (75 nM) relative to the two substrates (1.2 mM for the diamine and 4. 9 mM for the lactone) provides an advantage of 155 M for the bimolecular reaction and 1000 for the previously discussed cyclization reac tion. The observed ratio of katJkN (equivalent to kcatlkuncat) is 16 M and 790, in fair agreement with the theoretical estimates, which ignore the inadequacies of the transition-state mimic. Whereas the weaker-than-expected binding of transition-state analogs to enzymes has been attributed to either unfavor able steric interactions (a form of destabilization) or failure to form bind ing-site contacts (a form of unproductive binding) for antibodies, the transi tion-state analog acts as both model and mold. Thus the question of
Ab+L+A
1�
* KN
-
KLKA
Ab'L'A
* KAb
-
[LA1*+Ab
1�
-
P+Ab
KT
[AbLA1*
-
P+Ab
Figure 13 Reaction cycle to calculate the rate acceleration of the antibody-catalyzed reaction from the relative affinity of the antibody for the transition state versus the reactant (47).
40
BENKOVIC
fit is much less important than the accurate mimicry of the stereochemical and electronic characteristics of the transition state for the reaction in question. A compilation (86) of 18 KMIKi ratios, where KM is the Michaelis constant for substrate dissociation and Ki is the dissociation constant for the phosphon ate inhibitor for predominantly antibody-catalyzed ester hydrolysis (80-85), showed a linear though scattered correlation (slope 0.86, R2 0.80) with the ratio of kaJkN. The values of kab1kN range from 30 to 106 M and KMIKi from 4 to 3 X 104 M, (H20 at I M), with the former values roughly within one or two orders of magnitude of the latter (86). Since it is unlikely that the ratio of KMIKi will exceed 109 M (although this is more than respectable) , it is clear that rate-enhancements above this limit probably arise from the active participation of amino acid side-chains or added cofactors within the anti body-binding site. Positive deviations from this relationship, as we shall see, indeed are suggestive of more complex mechanistic processes. The transesterification reaction (Figure 5) is formally a bimolecular sub stitution reaction that like the aforementioned aminolysis reaction must com pete with the hydrolysis of the ester. Antibodies that previously had been induced with racemic phosphonate to catalyze the hydrolysis of the esters of R- and S- sec-phenethyl alcohols fell into two classes: a group that acted on the R- and a second set that acted on the S-substrates. One antibody, de signated 21H3, which was an S-specific esterase, also was found to catalyze the transesterification reaction in a mixture of water and 10% dimethyl sulfoxide through the two-step process shown in Figure 14. Remarkably, this antibody uses a ping-pong kinetic sequence, which is not limited to the vinyl ester but includes substituted benzylic esters as well. Compelling evidence adduced for the ping-pong s�quence includes: (a) dou ble reciprocal initial velocity plots that are a family of parallel lines; (b) inhibition patterns in which the ester product is a noncompetitive inhibitor of the alcohol substrate or the first alcohol product released competitively in hibits addition of the second alcohol; (c) the rapid, stoichiometric reaction of 21H3 with the p-nitrophenylester; and (d) the labeling of the antibody with a 14C radiolabel located in the acyl portion of the ester.
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=
o
21H3 +
21H3
)l + R
=
CH3CHO
0 H, CH3 o HO .H H3+ JlO R 21H3 )lR+ � CH3 � 21
V
whereR�
H CH3 N 'l( 'Y.". o
Figure 14
� V
�
Ping-pong kinetic sequence for transesterification reaction.
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CATALYTIC ANTmODIES
41
A measurement of the reaction rate in the absence of antibody was com plicated by hydrolytic cleavage of the ester at pHs at which sufficient alkoxide ion could be generated for observable transesterification. The comparison of kN and kab is further compromised by the fact that the mechanisms in the two cases are different, but with these caveats, a minimal estimate of lOs-106 M was obtained from the antibody-catalyzed rate for transesterification of the vinyl ester with sec-phenethyl alcohol. A common feature ascribed to protein conformation flexibility and often associated with enzymic catalysis is the induced fit of substrate to stabilize a rare, reactive conformer. The presence of sec-phenethyl alcohol provides a route for transesterification but also increases the esterase activity 1O-fold (J. Stewart, unpublished results). Similarly, surrogates devoid of an accepting hydroxyl, e.g. sec-phenethyl chloride and bromide, increase the rate of hydrolysis (VIK) of the vinyl ester by the antibody by a factor of 10. The simple induced-fit interpretation (88) is that in the absence of the surrogate, the antibody has either a conformation in which bound ester or the acyl intermediate is less accessible to water, or key transition states in which the acylationldeacylation pathway is less stabilized. The induction of an antibody capable of a ping-pong kinetic sequence would not at first glance appear to be implicit in the hapten design. But recall that these antibodies were produced in response to a monoesterified phosphonic acid derivative (Figure 6) with the intent to elicit esterase activity. If transesterification activity were the initial target, then the hapten should have been a diesterified phosphonate with either similar or dissimilar alcohol moieties. This would provide sufficient space in a programmed binding site for the simultaneous binding of both ester and alcohol to the antibody. One might argue, then, that in order for the transesterification reaction to be catalyzed by the present set of antibodies, it must follow a ping-pong se quence since the acceptor alcohol must occupy the same volume in space as the alcohol portion of the ester. What is remarkable then is the induction of a nucleophile at the binding site of the antibody in proper juxtaposition, with an appropriate pKa, and with a balanced acylation/deacylation reactivity to catalyze the transesterification reaction at such high efficiency.
Hydrolysis The antibody, 43C9, which catalyzes the hydrolysis of the p-nitroanilide (Figure 7) and a series of related aromatic esters (pNOz, pCI, pCH3, pCH3CO), has been the subject of an extensive number of experiments to establish its mechanism of action (89). The steady-state Michaelis-Menten parameters, kcatlKM and kcat> as a function of pH for both the p-nitroanilide and ester substrates, are exhibited in Figure 15. At high pH the observed kcat exceeds the ratio of K/KM by > lOO-fold. The data can be fit either to a reaction mechanism involving the titration of a group whose dissociation (pKa
42
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Figure 15 pH-rate profiles for the hydrolysis of the p-nitrophenylester (left panel) and p nitroanilide (right panel) by antibody, 43C9 (see Figure 7) (89). =
9.0) promotes substrate hydrolysis, or to a mechanism that features a
change in the rate-limiting step around an intermediate antibody-bound spe cies due to changes in pH. Support for the latter sequence stems from several lines of evidence. Firstly, that a true pKa is not being observed in the pH-rate profiles is in accord with the observations that >
(a) the plateau rate encountered in kcat at pH 9.0 for hydrolysis of the p-nitroester is a consequence of the slow dissocia
tion of the p-nitrophenol product; (b) the rate constants for binding of p nitrophenol as well as the dissociation constant Ki (for the p-nitroanilide as a competitive inhibitor of ester hydrolysis) are pH independent over the range of measurement ;
(c) slow dissociation of p-nitrophenol should perturb the pKa of a group in common to both profiles by ca. 0.7 pH units for the ester relative to the anilide (90). Secondly, measurement of the pH-kcat and kca/KM profiles for both the hydrolysis of the anilide and the p-CI ester in deuterium oxide revealed no deuterium solvent isotope effect on kcat or kca/KM at pH > 9.0 but a substantial effect (k2H20/k2D20 = 2-4) on kcat and kca/KM at pH < 9.0 (91, 92). These observations rule out a simple general base-catalyzed hydrolytic mechanism involving a single dissociable group, and are only in accord with two different rate processes defining the apparent pKa. Evidence for the presence of an acyl intermediate sought by both stopped flow and rapid quench methods did not reveal any accumulation above
(89, 92). However, the hydrolysis of a series 43C9 gave kcat values above pH 9.0 , which were
undetectable steady-state levels of aromatic esters by
strongly dependent on the electronic nature of the p-substituent. The Hammett p value is
2.3, consistent with values of 2-3 observed for attack on acyl esters
CATALYTIC ANTIBODIES
Figure 16
43
Kinetic reaction sequence for es
ter and anilide hydrolysis catalyzed by 43C9
Annu. Rev. Biochem. 1992.61:29-54. Downloaded from www.annualreviews.org by Syracuse University Library on 04/22/13. For personal use only.
(89).
by nitrogen nucleophiles (93) but not values of 1.0-1. 2 and 0.5-0.7 found for nucleophilic attack by oxyanions or general base catalysis (94). This observa tion, coupled with a lack of 180 exchange from H2 180 into unreacted anilide in the presence of antibody, also rules out the accumulation of a symmetrical tetrahedral intermediate. The weight of the data then favor the reaction sequence given in Figure 16, where S is either anilide or ester substrate, I is the acylated antibody plus P2, PI is the acid product, and P2 the phenol or aniline. The pH-rate profiles arise from a change in rate-limiting step, from product release (ester, kcat) or acylation (anilide, kcatlKM) at high pH, to deacylation (kcat and kcatlKM) at low pH. From the primary sequence of the antibody, 43C9, the most likely candidate for the nucleophile is a histidine located in the L3 loop of the light chain. However, the appearance of a large deuterium solvent isotope effect on the deacylation leg is not in accord with attack by hydroxide on an acyl intermediate, but suggests that a second basic group on the antibody may be acting to catalyze deacylation. Its pKa, like that of the nucleophile, is outside the range of the pH-rate profiles. The relative importance of the various kinetic steps in antibody turnover can be more easily visualized through the aid of a free energy (LlC*) reaction coordinate diagram (Figure 17). There are several striking features in the two profiles. The first is the general uneveness in the LlC* barriers for the various internal antibody-substrate complexes and their respective transition states. A more optimal situation would balance the differences in LlC:): between the external and internal ground states as well as their respective transition states-as is the case for enzymes operating at optimal flux-so that no single AC+ barrier would be egregious (95). A second is the high stability of the Ab·PI·P2 product complex relative to the respective uncomplexed substrates. It is this tight product complex that ultimately limits the rate of ester hydroly sis (kcat) and also is responsible for the lack of Ab·I accumulation. The distortion introduced by tight product binding has been remedied by chang ing the nature of the p-substituent, e.g. pN02 to pCl, but with a drop in the acylation rate (92). A third feature is the increased kinetic
44
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