Phosphonate Transition State Analogue Research Articles

Biomimetic recognition and peptidase activities of transition state analogue imprinted chymotrypsin mimics

The first peptidolysis reaction utilizing transition state analogue imprinted polymer was demonstrated utterly in the viewpoint of size and shape-selective substrate recognition. The enzyme mimic polymer was synthesized from the amino acid triad histidine, aspartic acid and serine in presence of phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate transition state analogue (TSA) by molecular imprinting technique. The polymer catalyst synthesized exhibited high selectivity and performed as a reliable tool for peptidolytic reactions. The peptidase activity of the enzyme mimic polymer catalyst was investigated by following the hydrolysis of dipeptides spectrophotometrically at 207nm and the kinetic parameters, rate acceleration kaccand imprinting efficiencykim, were evaluated. The imprinted peptidase displayed a rate acceleration of 1.67×103 contrasted with the uncatalyzed peptidolysis and an imprinting efficiency of 45 over the non-imprinted control polymer. The artificial peptidase amazingly promoted the hydrolysis of dipeptides having Phe/Tyr amino acid as the C-terminal residues discriminating chymotrypsin specific and non-specific substrates. The mimic exhibited higher rate acceleration and substrate specificity towards peptides compared to amino acid p-nitroanilides. Despite the fact that natural enzyme is much superior to the MIP catalyst in hydrolase activities, the mimic portrays high thermal stability, prolonged life span and unrivaled recyclability and reusability.

Structural basis of the broad substrate tolerance of the antibody 7B9-catalyzed hydrolysis of p-nitrobenzyl esters

Catalytic antibody 7B9, which was elicited against p-nitrobenzyl phosphonate transition-state analogue (TSA) 1, hydrolyzes a wide range of p-nitrobenzyl monoesters and thus shows broad substrate tolerance. To reveal the molecular basis of this substrate tolerance, the 7B9 Fab fragment complexed with p-nitrobenzyl ethylphosphonate 2 was crystallized and the three-dimensional structure was determined. The crystal structure showed that the strongly antigenic p-nitrobenzyl moiety occupied a relatively shallow antigen-combining site and therefore the alkyl moiety was located outside the pocket. These results support the observed broad substrate tolerance of 7B9 and help rationalize how 7B9 can catalyze various p-nitrobenzyl ester derivatives. The crystal structure also showed that three amino acid residues (AsnH33, SerH95, and ArgL96) were placed in key positions to form hydrogen bonds with the phosphonate oxygens of the transitions-state analogue. In addition, the role of these amino acid residues was examined by site-directed mutagenesis to alanine: all mutants (AsnH33Ala, SerH95Ala, and ArgL96Ala) showed no detectable catalytic activity. Coupling the findings from our structural studies with these mutagenesis results clarified the structural basis of the observed broad substrate tolerance of antibody 7B9-catalyzed hydrolyses. Our findings provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates, aiding their practical application in synthetic organic chemistry.

Catalytic amidolysis of amino acid p-nitroanilides using transition state analogue imprinted artificial enzymes: Cooperative effect of pyridine moiety

Enzyme-like polymer catalysts with the imprints of phosphonate transition state analogue (TSA) lined along with imidazole and pyridine moieties were synthesized using methacryloyl-l-histidine and 4-vinylpyridine as the functional monomers and phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate – the TSA of hydrolytic reaction as the template for the amidolysis of N-benzyloxycarbonyl-l-phenylalanine p-nitroanilide (Z-l-Phe-PNA). Polymers containing different functional groups can act together to provide catalytic activity and selectivity superior to what can be obtained from monofunctional analogues. The higher rate acceleration exhibited by the bifunctional polymer over the monofunctional polymers indicates cooperative catalysis of imidazole and pyridine moieties. The optimum catalytic competence is shown by the bifunctional polymer containing imidazole and pyridine moieties in 2:1M ratio which may be due to alignment of the functional groups in proper H-bond distance. In addition to the non-covalent interactions like hydrogen bonding or π-stacking interactions between the functional groups of the polymer and the template, 3D-microcavities complementary to the geometry of the template are necessary for effective shape selective binding. Michaelis-Menten kinetics implies that only the catalysts with imidazole moieties act as enzyme-like catalysts and imidazole is the key catalytic function of the enzyme mimics.

Selection of Chiral Zinc Catalysts for the Kinetic Resolution of Esters via Dynamic Templating

Dynamic combinatorial libraries of chiral tetradentate bis-imine zinc(II) complexes have been prepared and screened for (1) their discrimination of enantiomeric picolinate esters and pyridyl phosphonate transition state analogs (TSAs) and (2) their catalytic activity and selectivity for enantioselective methanolysis of racemic picolinate esters. The zinc complexes are in equilibrium with their imine ligands as well as with the aldehyde and amine building blocks that form them, enabling the composition of the library to adapt in response to the introduction of coordinating substrates or TSAs. Binary (L)Zn(OTf)(solv)(+) complexes are generated either individually or in libraries from chiral tartrate-derived diamines (2,3) and a set of N-heterocyclic aldehydes (4-12) and the distribution of complexes established by ESI-MS analysis. Binding studies of the (diimine)Zn(OTf)(2) complex libraries with enantiomeric R- and S-2-pyridyl phosphonate TSA 13 show chiral discrimination via formation of diastereomeric LZn(R/S-13)(+) complexes with low to moderate enatioselectivity ratios, k(R)/k(S) (α), ranging from 0.5 to 5.0; corresponding templating of selected binary complexes with the enantiomeric substrates, PyrCO(2)CH(OH)Ph (1), show negligible chiral recognition. The rate constants for methanolysis of the R- and S-esters, PyrCO(2)CH(OH)Ph (1) catalyzed by several L*Zn(OTf)(2) complexes range in value several fold depending on L and with enantioselectivity ratios, k(R)/k(S) (α), ranging from 0.76 to 2.8.

New chiral Schiff base–zinc complexes and their esterolytic catalytic activity

A new series of chiral Zn–Schiff base (imine) complexes has been prepared from mono- N-sulfonyl derivatives of (1R,2R)-diaminocyclohexane, N-heterocyclic aldehydes, and zinc salts. The formation and characterization of the (L)ZnX 2 complexes was established by NMR, IR and HRESI-MS. Spectroscopic and kinetic evidence indicates that these ligands may be bidentate or tridentate depending on conditions of the medium. The methanolysis of a chiral, racemic picolinate ester catalyzed by the Zn(II)–Schiff base complexes was studied kinetically. The rate constants were found to vary approximately a hundred-fold and in a complex way depending on the imine ligand and the Zn-counter anion, k obs = 5.0 × 10 −6–4.8 × 10 −4 M −1 s −1. A Job plot analysis of ternary complex formation of LZnX 2 with two phosphonate transition state analogs suggests that two types of (imine)Zn(picolinate) X ternary complexes may be intermediates and that varying rate-limiting steps may be involved in the LZnX 2-catalyzed methanolysis of picolinate esters.

Molecular Basis for Transition-State Stabilization in Catalytic Antibodies

Abstract Antibodies elicited against rationally designed transition-state analogs catalyze a large number of reactions, including many that cannot be achieved by standard chemical methods. This account describes a detailed investigation of the molecular mechanism of catalysis in catalytic antibodies. We have investigated the biochemical properties within a panel of six hydrolytic catalytic antibodies elicited against a phosphonate transition-state analog. The transition-state analyses (kcat⁄kuncat versus Km⁄Ki) displayed a linear relationship with five antibodies, 6D9, 8D11, 4D5, 3G6, and 9C10, which have homologous primary amino acid sequences, while antibody 7C8 deviates from the linear relationship. These results suggest that the five antibodies catalyze the hydrolysis by the same reaction mechanism of transition-state stabilization and that catalytic factors other than transition-state stabilization are involved for antibody 7C8. The structure of 6D9 Fab with the transition-state analog (TSA) has been solved, showing that 6D9 binds the phosphonate of TSA by forming one hydrogen bond with HisL27d. The kinetic, structural, and thermodynamic analyses of 6D9 indicate that this antibody provides an efficient catalyst by transition-state stabilization through the hydrogen bond with HisL27d and the destabilization of the substrate. Although catalytic antibodies are still much inferior to natural enzymes in their catalytic efficiency, several aspects including the mechanism of enzyme catalysts can be faithfully mimicked. Therefore, the molecular mechanism of catalytic antibodies might provide useful insights to understanding the catalysis in enzymes.

Adenosine phosphonate inhibitors of lipid II: Alanyl tRNA ligase MurM from Streptococcus pneumoniae

Adenosine and 2′-deoxyadenosine phosphonate transition state analogues act as the first inhibitors for the MurMN/FemABX family of tRNA-dependent ligases implicated in high-level penicillin resistance in Gram-positive bacteria.

Enzymatic resolution of hindered cyanohydrins, key precursors of muscarinic receptor antagonists

Enantiomerically pure 1-cycloalkyl-1-hydroxy-1-phenylcyanohydrins, key precursors in the synthesis of ( S)-oxybutynin and other antimuscarinic agents, have been successfully prepared via lipase-catalyzed kinetic resolutions. Pseudomonas cepacia and Candida antarctica lipase B have shown excellent enantioselectivities in hydrolysis and acylation processes, depending on the substrate structure and the reaction conditions. Furthermore, molecular modeling of phosphonate transition state analogue for the C. antarctica lipase B enzymatic hydrolysis resolution step supports these facts, which were experimentally observed.

Identification and Characterization of Single Chain Anti-cocaine Catalytic Antibodies

Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis.

X-ray structure of a bound phosphonate transition state analog and enantioselectivity ofCandida rugosalipase toward chiral carboxylic acids

X-ray structure of a bound phosphonate transition state analog and enantioselectivity of<i>Candida rugosa</i>lipase toward chiral carboxylic acids

An Inverse Substrate Orientation for the Regioselective Acylation of 3′,5′‐Diaminonucleosides Catalyzed by Candida antarctica lipase B?

Candida antarctica lipase B (CAL-B) catalyzes the regioselective acylation of natural thymidine with oxime esters and also the regioselective acylation of an analogue, 3',5'-diamino-3',5'-dideoxythymidine with nonactivated esters. In both cases, acylation favors the less hindered 5'-position over the 3'-position by upto 80-fold. Computer modeling of phosphonate transition-state analogues for the acylation of thymidine suggests that CAL-B favors acylation of the 5'-position because this orientation allows the thymine ring to bind in a hydrophobic pocket and forms stronger key hydrogen bonds than acylation of the 3'-position. On the other hand, computer modeling of phosphonamidate analogues of the transition states for acylation of either the 3'- or 5'-amino groups in 3',5'-diamino-3',5'-dideoxythymidine shows similar orientations and hydrogen bonds and, thus, does not explain the high regioselectivity. However, computer modeling of inverse structures, in which the acyl chain binds in the nucleophile pocket and vice versa, does rationalize the observed regioselectivity. The inverse structures fit the 5'-, but not the 3'-intermediate thymine ring, into the hydrophobic pocket, and form a weak new hydrogen bond between the O-2 carbonyl atom of the thymine and the nucleophile amine only for the 5'-intermediate. A water molecule might transfer a proton from the ammonium group to the active-site histidine. As a test of this inverse orientation, we compared the acylation of thymidine and 3',5'-diamino-3',5'-dideoxythymidine with butyryl acyl donors and with isosteric methoxyacetyl acyl donors. Both acyl donors reacted at equal rates with thymidine, but the methoxyacetyl acyl donor reacted four times faster than the butyryl acyl donor with 3',5'-diamino-3',5'-dideoxythymidine. This faster rate is consistent with an inverse orientation for 3',5'-diamino-3',5'-dideoxythymidine, in which the ether oxygen atom of the methoxyacetyl group can form a similar hydrogen bond to the nucleophilic amine. This combination of modeling and experiments suggests that such lipase-catalyzed reactions of apparently close substrate analogues like alcohols and amines might follow different pathways.

Unusual Four-Bond Secondary H/D Isotope Effect Supports a Short−Strong Hydrogen Bond between Phospholipase A2 and a Transition State Analogue Inhibitor

A prominent secondary four-bond hydrogen/deuterium isotope effect was observed from proton NMR at the active site histidine imidazole ring of bovine pancreatic sPLA(2) in the presence of a phosphonate transition state analogue. The cross-modulation of H(epsilon2)/H48 and H(delta1)/H48 resonances was confirmed by line shape simulation that follows the McConnell equation with fractionation factors incorporated to account for the change in the signal magnitude as well as the resonance line shape at various H(2)O/D(2)O solvent mixtures. While the downfield shift of each individual proton upon deuteration on the opposite site can be attributed to the proton-relay system of the H48-D99 catalytic dyad in sPLA(2), the observation that H(delta1)/H48 induces a 3-fold larger H/D secondary isotope effect ( approximately 0.15 ppm) on H(epsilon2)/H48 than vice versa ( approximately 0.05 ppm) is interpreted as additional spectroscopic evidence for the previously proposed short-strong hydrogen bond formed between the donor N(delta1)/H48 and a nonbridging phosphonate oxygen atom of the transition state analogue. These results provide additional details for the catalytic mechanism of sPLA(2) and demonstrate that the intrinsic H/D secondary isotope effect is a useful tool to probe hydrogen bond strength.

Antibody-catalyzed formation of a 14-membered ring lactone

Monoclonal antibody (MAb) F123, raised against a macrocyclic phosphonate transition-state analogue, catalyzed an intramolecular transesterification of the corresponding hydroxy ester to give a 14-membered ring lactone. The MAb reaction displayed enzyme-like Michaelis–Menten kinetics with a Km of 255 µM and a kcat of 0.01 min–1 based on p-nitrophenol release and calculated on an active-site basis. Substrate specificity and competitive inhibition by a transition state analogue (Ki = 3 µM) demonstrated that the catalytic activity was associated with binding in the antibody-combining site. The lactone product was isolated from a large-scale catalytic experiment through ether extraction and identified by gas chromatography – mass spectroscopy.Key words: macrocyclization, lactones, catalytic antibodies.

Selective chemotherapeutic strategies using catalytic antibodies: a common pro-moiety for antibody-directed abzyme prodrug therapy

Prodrug activation by catalytic antibodies (abzymes) conjugated with anti-tumor antibodies, called antibody-directed abzyme prodrug therapy (ADAPT), has been proposed as a strategy for site-specific drug delivery systems for anti-tumor drugs. The delivery of abzymes is achieved by making a bi-specific antibody with a monovalent catalytic antibody and a monovalent binding antibody. To achieve ADAPT, we focused on specific requirements for prodrugs and catalytic antibodies, the stability of the prodrugs against natural enzymes, and the applicability of abzymes for a wide range of prodrugs. Attention was paid to the design of a pro-moiety rather than a parent drug. As a common pro-moiety, we chose vitamin B 6, because the bulky vitamin B 6 esters are relatively stable against hydrolytic enzymes in serum. We have generated catalytic antibodies by immunization of a vitamin B 6 phosphonate transition state analog. The elicited antibodies were found to hydrolyze several anti-cancer and anti-inflammatory prodrugs with the vitamin B 6 pro-moiety. Finally, we evaluated antibody-catalyzed prodrug activation by examining the growth inhibition of human cervical cancer (HeLa) cells with the vitamin B 6 ester of butyric acid. These results suggest that the pro-moiety of vitamin B 6 ester is stable enough to resist natural enzymes in serum and is removed by the tailored catalytic antibodies. The combination of catalytic antibodies and prodrugs masked with vitamin B 6 would allow hydrophobic and highly toxic drugs to be used.

Molecular Evolution of Catalytic Antibodies in Autoimmune Mice

Catalytic Abs (catAbs) preferentially evolved in autoimmune MRL/MPJ-lpr/lpr (MRL/lpr) mice upon immunization with the phosphonate transition-state analogue (TSA), but this did not happen in normal BALB/c mice. The majority of the catAbs from MRL/lpr mice were from several independent clones of the same family. Most of them had a lysine at position 95 in the heavy chain (H95), which is at the junctional region. This residue, which interacts with the phosphonate moiety of the TSA and presumably is involved in the catalytic activity, was not changed even after expansive evolution following multiple mutations. By contrast, the majority that arose from BALB/c mice were the non-catAbs, which were quite different in the sequence from the catAbs from MRL/lpr mice, but they were clonally related to one another, so most of them were originated from a single clone. In the MRL/lpr mice, the catalytic subsets that existed in the initial repertoire were effectively captured by the phosphonyl oxygens in the TSA by interacting with the lysine at H95. In the BALB/c mice, however, another noncatalytic subset with only the binding capability directed to a moiety other than the phosphonate moiety was alternatively evolved, because of the lowest abundance or elimination of the catalytic subsets.

Molecular basis for enantioselectivity of lipase from Chromobacterium viscosum toward the diesters of 2,3-dihydro-3-(4'-hydroxyphenyl)-1,1,3-trimethyl-1H-inden-5-ol.

2,3-Dihydro-3-(4'-hydroxyphenyl)-1,1,3-trimethyl-1H-inden-5-ol, 1, is a chiral bisphenol useful for preparation of polymers. Previous screening of commercial hydrolases identified lipase from Chromobacterium viscosum (CVL) as a highly regio- and enantioselective catalyst for hydrolysis of diesters of 1. The regioselectivity was > or =30:1 favoring the ester at the 5-position, while the enantioselectivity varied with acyl chain length, showing the highest enantioselectivity (E = 48 +/- 20 S) for the dibutanoate ester. In this paper, we use a combination of nonsymmetrical diesters and computer modeling to identify that the remote ester group controls the enantioselectivity. First, we prepared nonsymmetrical diesters of (+/-)-1 using another regioselective, but nonenantioselective, reaction. Lipase from Candida rugosa (CRL) showed the opposite regioselectivity (>30:1), allowing removal of the ester at the 4'-position (the remote ester in the CVL-catalyzed reaction). Regioselective hydrolysis of (+/-)-1-dibutanoate (150 g) gave (+/-)-1-5-dibutanoate (89 g, 71% yield). Acylation gave nonsymmetrical diesters that varied at the 4'-position. With no ester at the 4'-position, CVL showed no enantioselectivity, while hindered esters (3,3-dimethylbutanoate) reacted 20 times more slowly, but retained enantioselectivity (E = 22). These results indicate that the remote ester group can control the enantioselectivity. Computer modeling confirmed these results and provided molecular details. A model of a phosphonate transition state analogue fit easily in the active site of the open conformation of CVL. A large hydrophobic pocket tilts to one side above the catalytic machinery. The tilt permits the remote ester at the 4'-position of only the (S)-enantiomer to bind in this pocket. The butanoate ester fits and fills this pocket and shows high enantioselectivity. Both smaller and larger ester groups show low enantioselectivity because small ester groups cannot fill this pocket, while longer ester groups extend beyond the pocket. An improved large-scale resolution of 1-dibutanoate with CVL gave (R)-(+)-1-dibutanoate (269 g, 47% yield, 92% ee) and (S)-(-)-1-4'-monobutanoate (245 g, 52% yield, 89% ee). Methanolysis yielded (R)-(+)-1 (169 g, 40% overall yield, >97% ee) and (S)-(-)-1 (122 g, 36% overall yield, >96% ee).

A Predictive Approach Using Fractal Analysis for Analyte–Receptor Binding and Dissociation Kinetics for Surface Plasmon Resonance Biosensor Applications

A predictive approach using fractal analysis is presented for analyte–receptor binding and dissociation kinetics for biosensor applications. Data taken from the literature may be modeled, in the case of binding using a single-fractal analysis or a dual-fractal analysis. The dual-fractal analysis represents a change in the binding mechanism as the reaction progresses on the surface. A single-fractal analysis is adequate to model the dissociation kinetics in the examples presented. Predictive relationships developed for the binding and the affinity (kdiss/kbind) as a function of the analyte concentration are of particular value since they provide a means by which the binding and the affinity rate coefficients may be manipulated. Relationships are also presented for the binding and the dissociation rate coefficients and for the affinity as a function of their corresponding fractal dimension, Df, or the degree of heterogeneity that exists on the surface. When analyte–receptor binding or dissociation is involved, an increase in the heterogeneity on the surface (increase in Df) leads to an increase in the binding and in the dissociation rate coefficient. It is suggested that an increase in the degree of heterogeneity on the surface leads to an increase in the turbulence on the surface owing to the irregularities on the surface. This turbulence promotes mixing, minimizes diffusional limitations, and leads subsequently to an increase in the binding and in the dissociation rate coefficient. The binding and the dissociation rate coefficients are rather sensitive to the degree of heterogeneity, Df,bind (or Df1) and Df,diss, respectively, that exists on the biosensor surface. For example, the order of dependence on Df,bind (or Df1) and Df2 is 6.69 and 6.96 for kbind,1 (or k1) and k2, respectively, for the binding of 0.085 to 0.339 μM Fab fragment 48G7L48G7H in solution to p-nitrophenyl phosphonate (PNP) transition state analogue immobilized on a surface plasmon resonance (SPR) biosensor. The order of dependence on Df,diss (or Df,d) is 3.26 for the dissociation rate coefficient, kdiss, for the dissociation of the 48G7L48G7H–PNP complex from the SPR surface to the solution. The predictive relationships presented for the binding and the affinity as a function of the analyte concentration in solution provide further physical insights into the reactions on the surface and should assist in enhancing SPR biosensor performance. In general, the technique is applicable to other reactions occurring on different types of biosensor surfaces and other surfaces such as cell-surface reactions.

Remote Binding Energy in Antibody Catalysis: Studies of a Catalytically Unoptimized Specificity Pocket

Binding interactions remote from the hydrolytic reaction center have been probed with substrate and phosphonate transition state analogues to understand how these types of interactions are used to promote catalysis in the 17E8 system. We find that the hapten-generated recogniton pocket in 17E8 has properties that are analogous to those of specificity pockets in enzymes. We have also found that there are specific requirements to form catalytically productive interactions between the side chain and the recognition pocket including conformation, size, and geometry. An additional requirement includes favorable simultaneous interactions between the side chain and binding pocket along with favorable interactions with the oxyanion hole. The 17E8 side chain recognition pocket seems to be less catalytically efficient than analogous pockets in enzymatic systems. The apparent binding energy gained from the methylene−pocket interactions in the 17E8 system is significantly smaller than those observed in natural enzyme...

Comparison of phosphonate transition state analogs for inducing catalytic antibodies and evaluation of key structural factors by an ab initio study

The relation between the structure of haptens and the esterolytic activities of antibodies was investigated. We synthesized two phenylalanine analogs, the negatively charged phosphonate derivative 1 and the neutral phosphonamidate derivative 2. Seventeen out of 41 monoclonal antibodies generated against the hapten 1 hydrolyzed the relevant phenylalanine ester R- 12 . On the contrary, none of 27 monoclonal antibodies generated against the hapten 2 had catalytic activity. An ab initio study of the structural and electronic properties of the modeled haptens showed that the value of the negative electrostatic potential around the phosphonyl oxygen was an important factor affecting the induction of esterolytic antibodies.

Conformational changes affect binding and catalysis by ester-hydrolysing antibodies

D2.3, D2.4 and D2.5 are ester-hydrolysing antibodies raised against a phosphonate transition state analogue (TSA). All three antibody-TSA binding kinetics, as monitored by fluorescence quenching, indicate an “induced-fit” mechanism: fast bimolecular association followed by a unimolecular isomerisation (k = 1-7 s−1). Isomerisation leads to a 30-170-fold increase in affinity towards the TSA and, consequently, to higher catalytic rates. Antibody D2.3 exhibits a complex three-step binding mechanism, in which the last step is a “very slow” isomerisation (k < 0.02 s−1). This very slow isomerisation is limiting the rate of catalysis by D2.3, as indicated by the kinetics of product release which show characteristics of enzyme “conformational memory” or “hysteresis”. The results support a mechanism consisting of pre-equilibrium between “nether-active” (low affinity) and “active” (high affinity) antibody conformers (prior to ligand addition) as well as induced-fit, i.e. isomerisation of the nether-active ligand-antibody complex to give the active complex. Crystal structures of these antibodies, free and complexed, have previously indicated that their conformation does not change upon binding. Here, we show that the buffer used to crystallise the antibodies, and in particular its polyethylene glycol component, alters the pre-equilibrium in favour of the active conformer, leading to its crystallisation both in the presence and in the absence of the TSA.