Phosphonate Transition State Analogue Research Articles

A chemiluminescent catalytic antibody†

A monoclonal antibody elicited against a phosphonate transition state analogue was shown to initiate chemiluminescence from luminogenic spiro[adamantane-2,3′-(1,2-dioxetane)] substrates by selectively cleaving a benzoate ester triggering group.

Structures of Esterase-Like Catalytic Antibodies

We report structures of complexes of two esterase-like catalytic antibodies elicited against phosphonate transition-state analogues (TSAs) of the reaction they catalyse; the complexes studied are with these TSAs and species along the reaction pathway. The TSAs are deeply sequestered in a pocket located in the combining site at the interface of the two antibody polypeptide chains; the deepest part of this pocket is almost completely hydrophobic. The larger number and stronger hydrogen bonds between the antibodies and the TSAs as compared to those established with the corresponding substrates account for the catalytic activity; transition state stabilisation is through oxyanion holes elicited in response to the negatively charged phosphonate oxygens.

Structures of Esterase-Like Catalytic Antibodies

We report structures of complexes of two esterase-like catalytic antibodies elicited against phosphonate transition-state analogues (TSAs) of the reaction they catalyse; the complexes studied are with these TSAs and species along the reaction pathway. The TSAs are deeply sequestered in a pocket located in the combining site at the interface of the two antibody polypeptide chains; the deepest part of this pocket is almost completely hydrophobic. The larger number and stronger hydrogen bonds between the antibodies and the TSAs as compared to those established with the corresponding substrates account for the catalytic activity; transition state stabilisation is through oxyanion holes elicited in response to the negatively charged phosphonate oxygens.

Crossreactivity, efficiency and catalytic specificity of an esterase-like antibody

The antibody D2.3 catalyzes the hydrolysis of several p-nitrobenzyl and p-nitrophenyl esters with significant rate enhancement; product inhibition is observed with the former compounds but not with the latter. Whereas enzyme specificity has been extensively studied by X-ray crystallography, structural data on catalytic antibodies have thus far related only to one of the reactions they catalyze. To investigate the substrate specificity and the substrate relative to product selectivity of D2.3, we have determined the structures of its complexes with two p-nitrophenyl phosphonate transition state analogs (TSAs) and with the reaction product, p-nitrophenol. The complexes with these TSAs, determined at 1.9 Å resolution, and that with p-nitrobenzyl phosphonate determined previously, differ mainly by the locations and conformations of the ligands. Taken together with kinetic data, the structures suggest that a hydrogen bond to an atom of the substrate distant by eight covalent bonds from the carbonyl group of the hydrolyzed ester bond contributes to catalytic efficiency and substrate specificity. The structure of Fab D2.3 complexed with p-nitrophenol was determined at 2.1 Å resolution. Release of p-nitrophenol is facilitated due to the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate with the hydrophobic cavity where it is located, and to the absence of a direct hydrogen bond between the product and the Fab. Catalytic specificity and the manner of product release are both affected by interactions with substrate atoms remote from the reaction center that were not programmed in the design of the TSA used to elicit this antibody. Selection of a catalytic antibody that makes use of TSA unprogrammed features has been made practical because of the screening for catalytic efficiency incorporated in the procedure used to obtain it.

A comparison of the crystallographic structures of two catalytic antibodies with esterase activity

The crystallographic structure of the Fab fragment of the catalytic antibody, 29G11, complexed with an (S)-norleucine phenyl phosphonate transition state analog was determined at 2.2 Å resolution. The antibody catalyzes the hydrolysis of norleucine phenyl ester with (S)-enantioselectivity. The shape and charge complementarity of the binding pocket for the hapten account for the preferential binding of the (S)-enantiomer of the substrate. The structure is compared to that of the more catalytically efficient antibody, 17E8, induced by the same hapten transition state analog. 29G11 has different residues from 17E8 at eight positions in the heavy chain, including four substitutions in the hapten-binding pocket: A33V, S95G, S99R and Y100 AN, and four substitutions at positions remote from the catalytic site, I28T, R40K, V65G and F91L. The two antibodies show large differences in the orientations of their variable and constant domains, reflected by a 32° difference in their elbow angles. The V Land V Hdomains in the two antibodies differ by a rotation of 8.8°. The hapten binds in similar orientations and locations in 29G11 and 17E8, which appear to have catalytic groups in common, though the changes in the association of the variable domains affect the precise positioning of residues in the hapten-binding pocket.

A structural basis for transition-state stabilization in antibody-catalyzed hydrolysis: crystal structures of an abzyme at 1.8 Å resolution

The three-dimensional structure of a catalytic antibody, 6D9, has been solved as a complex with a transition state analog. The structure was determined from two different crystal forms, and was refined at a resolution of 1.8 Å. The antibody 6D9, which was induced by immunization with the phosphonate transition state analog 3, hydrolyzes a prodrug of chloramphenicol monoester 1 to generate the parent drug 2. The kinetic studies have shown that the antibody is catalytic by virtue of the theoretical relationship between the affinity for the transition state and the catalytic efficiency ( k cat/ k uncat= K S/ K TSA). The crystal structure makes it possible to visualize the theoretical relationship. A side-chain (N ϵ) of His L27Dis placed in a key position to make a hydrogen bond to the phosphonate oxygen of the transition state analog with a distance of 2.72 Å, suggesting a hydrogen bond to the oxyanion developing in the transition state of the hydrolysis. There are no catalytic residues, other than the histidine, around the phosphonate moiety. In addition, in the antibody-hapten complex, the hapten bears a folded conformation and the two stacked aromatic rings are buried deep in the antigen-combining site through aromatic-aromatic interaction with Trp H100Iand Tyr H58. The conformation of the bound hapten suggests that the antibody binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable E-form, thereby making it easy for the substrate to reach the transition-state during catalysis. These observations reveal that the catalytic mechanism is explained purely on the basis of the stabilization of the transition state. The refined high resolution structures reported here are envisaged to have an impact on the understanding of other hydrolytic antibodies, since their haptens share some unique features with the hapten used in this study.

Similarities of hydrolytic antibodies revealed by their X-ray structures: A review

Numerous antibodies have been programmed to catalyse the hydrolysis of esters as well as other acyl transfer reactions. They were raised against stable analogues that model the structure of the tetrahedral transition states of these reactions. The three-dimensional structures of four hydrolytic antibodies complexed to their respective phosphonate transition state analogues (TSAs) reveal a similar orientation of hapten relative to the antibody. Analysis of the four combining sites suggests that residues binding the phosphonate TSA stabilise the oxyanion intermediate of the reaction and play a preponderant role in catalysis. Comparison of catalytic antibodies selected from the same hybridoma fusion indicates a high similarity of the motif that catalyse the hydrolysis of a given substrate.

Phage display of a catalytic antibody to optimize affinity for transition-state analog binding.

Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. "Humanized" 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100aH --> Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.

X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release.

The x-ray structures of the unliganded esterase-like catalytic antibody D2.3 and its complexes with a substrate analogue and with one of the reaction products are analyzed. Together with the structure of the phosphonate transition state analogue hapten complex, these crystal structures provide a complete description of the reaction pathway. At alkaline pH, D2.3 acts by preferential stabilization of the negatively charged oxyanion intermediate of the reaction that results from hydroxide attack on the substrate. A tyrosine residue plays a crucial role in catalysis: it activates the ester substrate and, together with an asparagine, it stabilizes the oxyanion intermediate. A canal allows facile diffusion of water molecules to the reaction center that is deeply buried in the structure. Residues bordering this canal provide targets for mutagenesis to introduce a general base in the vicinity of the reaction center.

Efficient and selective p-nitrophenyl-ester-hydrolyzing antibodies elicited by a p-nitrobenzyl phosphonate hapten.

A number of monoclonal antibodies elicited against a nitrobenzyl (Nbzl)-phosphonate transition-state analogue (TSA), and which were selected for the hydrolysis of the corresponding Nbzl-ester, were also found to catalyze the hydrolysis of the analogous p-nitrophenyl(Np) ester with notable efficiency and specificity. The activity towards the Np-ester is higher in terms of rates (k(cat); as expected from the higher intrinsic reactivity of Np-esters); however, the rate acceleration (k(cat)/k(uncat)) is close to or lower than that observed with the Nbzl-ester. Unexpectedly, the affinity to the Np-ester substrate (1/K(M)) and therefore k(cat)/K(M) are significantly higher. The best example is antibody D2.4 having a k(cat)/K(M) value of 64 s(-1) x M(-1) with the Nbzl-ester and 9400 s(-1) x M(-1) with the Np-ester. Moreover, due to a lower product inhibition by p-nitrophenol relative to p-nitrobenzyl alcohol, these antibodies exhibit more than 1000 turnovers with the Np-ester. The differential affinity of these antibodies to the Nbzl-phosphonate TSA versus the Nbzl-ester substrate (K(S)/K(TSA) or K(M)/K(i)) correlates well with the observed rate enhancement (k(cat)/k(uncat)). For the Np-ester, however, stabilisation of the transition state (as reflected by K(S)/K(TSA) and by the catalytic proficiencies, k(cat)/K(M)/k(uncat)) does not fully account for the catalytic power (k(cat)/k(uncat)), indicating a more complex catalytic mechanism than simply transition-state stabilization. A comparison of the kinetic parameters of D2.4 with other Np-ester-hydrolyzing antibodies raised against Np-phosphonate haptens emphasizes the marked advantage of this antibody which was elicited against an Nbzl-phosphonate hapten. These results appear to be general: anti-(Nbzl-phosphonate TSA) antibodies obtained from other mouse strains and using different immunization protocols are also efficient Np-esterases. They demonstrate the use of an expanded TSA-hapten, where a spacer (a methylene group) mimics bonds that are partially cleaved in the transition state of the catalyzed reaction.

Structural convergence in the active sites of a family of catalytic antibodies.

The x-ray structures of three esterase-like catalytic antibodies identified by screening for catalytic activity the entire hybridoma repertoire, elicited in response to a phosphonate transition state analog (TSA) hapten, were analyzed. The high resolution structures account for catalysis by transition state stabilization, and in all three antibodies a tyrosine residue participates in the oxyanion hole. Despite significant conformational differences in their combining sites, the three antibodies, which are the most efficient among those elicited, achieve catalysis in essentially the same mode, suggesting that evolution for binding to a single TSA followed by screening for catalysis lead to antibodies with structural convergence.

Transition State Analysis Within a Panel of Catalytic Antibodies Generated Against a Phosphonate Transition State Analog

Abstract Preparation of phosphinic chlorides, amides, and sulfide derivatives, as well as phosphine-borane complexes of P-heterocycles is described.

Transition State Analysis within a Panel of Catalytic Antibodies Generated Against a Phosphonate Transition State Analog

Transition State Analysis within a Panel of Catalytic Antibodies Generated Against a Phosphonate Transition State Analog

P-A5-01 Phage display selection of glutathione transferase A1-1 mutants with esterase activity, using their affinity for a phosphonate transition-state analog: Nilsson L. O., Widersten M., Mannervik B. Dept. of Biochemistry, Uppsala University (S)

P-A5-01 Phage display selection of glutathione transferase A1-1 mutants with esterase activity, using their affinity for a phosphonate transition-state analog: Nilsson L. O., Widersten M., Mannervik B. Dept. of Biochemistry, Uppsala University (S)

Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies.

The x-ray structure of the complex of a catalytic antibody Fab fragment with a phosphonate transition-state analog has been determined. The antibody (CNJ206) catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and substrate specificity. Comparison of this structure with that of the uncomplexed Fab fragment suggests hapten-induced conformational changes: the shape of the combining site changes from a shallow groove in the uncomplexed Fab to a deep pocket where the hapten is buried. Three hydrogen-bond donors appear to stabilize the charged phosphonate group of the hapten: two NH groups of the heavy (H) chain complementarity-determining region 3 (H3 CDR) polypeptide chain and the side-chain of histidine-H35 in the H chain (His-H35) in the H1 CDR. The combining site shows striking structural similarities to that of antibody 17E8, which also has esterase activity. Both catalytic antibody ("abzyme") structures suggest that oxyanion stabilization plays a significant role in their rate acceleration. Additional catalytic groups that improve efficiency are not necessarily induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten.

Crystal Structure of a Catalytic Antibody with a Serine Protease Active Site

The three-dimensional structure of an unusually active hydrolytic antibody with a phosphonate transition state analog (hapten) bound to the active site has been solved to 2.5 A resolution. The antibody (17E8) catalyzes the hydrolysis of norleucine and methionine phenyl esters and is selective for amino acid esters that have the natural alpha-carbon L configuration. A plot of the pH-dependence of the antibody-catalyzed reaction is bell-shaped with an activity maximum at pH 9.5; experiments on mechanism lend support to the formation of a covalent acyl-antibody intermediate. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases. The antibody active site contains a Ser-His dyad structure proximal to the phosphorous atom of the bound hapten that resembles two of the three components of the Ser-His-Asp catalytic triad of serine proteases. The antibody active site also contains a Lys residue to stabilize oxyanion formation, and a hydrophobic binding pocket for specific substrate recognition of norleucine and methionine side chains. The structure identifies active site residues that mediate catalysis and suggests specific mutations that may improve the catalytic efficiency of the antibody. This high resolution structure of a catalytic antibody-hapten complex shows that antibodies can converge on active site structures that have arisen through natural enzyme evolution.

Effect of polar head groups on the interactions of phospholipase A2 with phosphonate transition-state analogues.

Several new phosphonate-containing phospholipid analogues were synthesized as inhibitors of cobra venom (Naja naja naja) phospholipase A2. These phospholipid analogues contained a novel thioether at the sn-1 position, a tetrahedral phosphonate moiety in place of the scissile ester bond at the sn-2 position, and several different polar head groups, including phosphocholine, phospho(N,N-dimethylethanolamine), phospho(N-methylethanolamine), and phosphoethanolamine. The affinities of these analogues for the enzyme were evaluated in the well-defined Triton X-100 mixed micelle system using thio-PC and thio-PE substrates. These phosphonates inhibited thio-PC hydrolysis with very similar potencies. Inhibition of phospholipase A2 by phosphonates is known to be pH-dependent [Yu, L., & Dennis, E. A. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9325-9329]. At pH 5.5, all of the new analogues had IC50s of about 2 x 10(-5) mol fraction. At this pH, these inhibitors are the most potent reversible inhibitors of phospholipase A2 reported to date. In contrast, at pH 8.5, the PE analogue was a potent inhibitor of thio-PC hydrolysis (IC50 1.8 x 10(-3) mol fraction) but was a very poor inhibitor of thio-PE hydrolysis (IC50 is not detectable). However, the inhibition of thio-PE hydrolysis was dramatically enhanced when the enzyme was activated by sphingomyelin, suggesting that the phosphonate inhibitors bind much more tightly to the activated enzyme than to the nonactivated enzyme. The activation and inhibition of the enzyme have different pH dependencies; the enzyme activation is not pH-dependent, whereas the enzyme inhibition is pH-dependent. These results confirm the presence of a functionally distinct activator site on this enzyme.

Prodrug activation via catalytic antibodies.

Prodrug activation via antibodies was examined by using the antibiotic chloramphenicol as a model drug. Based on the conformational change between substrate and product, this antibody-catalyzed reaction was designed to prevent product inhibition, thus enhancing turnover. Antibodies elicited against a phosphonate transition-state analogue were found to catalyze hydrolysis of a nonbioactive chloramphenicol monoester as a prodrug at a significantly higher rate above the uncatalyzed background reaction to regenerate chloramphenicol as a parent molecule. The antibody-catalyzed prodrug activation was tested by the paper-disc diffusion method using Bacillus subtilis as an indicator strain. The antibody 6D9 catalyzes the reaction with multiple turnover to generate enough chloramphenicol to inhibit bacterial growth, as indicated by a clear inhibitory zone after incubation with monoester. Using the same method, no inhibition was detected by incubation of either the monoester or the antibody alone. This result reveals that only the antibody hydrolytically activates the monoester, which can be expected to be a suitable prodrug, as it is resistant to the action of bacterial hydrolytic enzymes. The approach in this study demonstrates the use of catalytic antibody technology in medicine and may be applicable to drugs with undesirable effects, particularly in the field of cancer therapy.

A genetic approach to the generation of antibodies with enhanced catalytic activities.

A hydrolytic catalytic antibody, generated against a nitrophenyl phosphonate transition state analogue, has been cloned and expressed in Escherichia coli for use as a model system to demonstrate the feasibility of using genetic selections to enhance catalytic activity. Conditions were found that permit the secretion of active recombinant antibody into the periplasm of a strain of E. coli deficient in the biotin biosynthetic genes (delta bio-gal). A number of substrates were synthesized that, upon hydrolysis by the antibody, yield free biotin, which is required for cell growth. The substrates and selections can be used to identify mutants of the antibody with altered activities. This approach should be generalizable to a wide number of hydrolytic reactions including the selective cleavage of peptide, polysaccharide, phosphodiester, and ester bonds.

Critical role of a hydrogen bond in the interaction of phospholipase A2 with transition-state and substrate analogues.

The inhibition of phospholipase A2 by an amide substrate analogue, 1-hexadecylthio-2-hexadecanoyl-amino-1,2-dideoxy-sn-glycero-3-phos phocholine, and a phosphonate transition-state analogue, 1-hexadecylthio-1-deoxy-2-hexadecylphosphono-sn-glycero-3-ph osphocholine, is dramatically influenced by pH. However, these two inhibitors show opposite pH dependencies. The amide analogue acts more potently under basic conditions, whereas the phosphonate acts more potently under acidic conditions. In both cases, ligand binding is perturbed by protonation of an enzyme functional group with an apparent pKa of 6.1, which corresponds to that of a histidine residue. Thus, His-48, which has previously been implicated in catalysis, appears to be critically involved in the hydrogen bond interactions between the enzyme and these two inhibitors. The amide analogue binds most effectively to the enzyme when His-48 is deprotonated. Upon protonation of the histidine residue, the amide cannot form a critical hydrogen bond and loses its ability to interact effectively with the enzyme. In contrast, the phosphonate analogue binds much tighter to the protonated form of the enzyme than to the deprotonated form. The phosphonate analogue needs a bridging hydrogen between the oxygen on its phosphorus atom and the N delta 1 of His-48 to form a strong hydrogen bond. At optimal pH values for inhibitor binding, both the amide and the phosphonate analogues are potent competitive inhibitors of cobra (Naja naja naja) venom phospholipase A2. The IC50 for the amide was 4.4 x 10(-4) mol fraction and for the phosphonate was 1.6 x 10(-5) mol fraction. Under the experimental conditions used, this corresponds to a bulk concentration of 2 microM and 70 nM, respectively.