An investigation of antibody acyl hydrolysis catalysis using a large set of related haptens

An aspect of catalytic antibody research that receives little attention in the literature involves hapten systems that fail to elicit antibody catalysts despite a high affinity immune response and hapten designs that resemble those known to elicit catalysts. We have investigated a series of 12 phosphate and phosphonate haptens in a total of three animal systems. Dramatic and reproducible differences were observed in the catalytic activities of polyclonal antibodies elicited by the different haptens. A phosphate hapten with a phenyl ring on the side of the hapten opposite the linker elicited reproducibly high levels of polyclonal antibody catalytic activity. The other 11 haptens, most with benzyl groups on the side of the hapten opposite the linker, elicited immune responses in which catalytic activity was significantly weaker in terms of the level of observed catalytic activity, as well as frequency of elicited catalysts. Our results indicate that subtle features of transition state analogue hapten structure can have a dramatic and reproducible influence over the catalytic activity of elicited antibodies in related haptens. Whatever the explanation, subtle changes in mechanistic features due to altered leaving group ability/location or overall hapten flexibility, the comprehensive data presented here indicate that phenyl or 4-nitrophenyl leaving groups located opposite the hapten linker are to be preferred in order to elicit highly active antibody catalysts for acyl hydrolysis reactions.     

Theory of equilibrium binding of symmetric bivalent haptens to cell surface antibody: application to histamine release from basophils.

We present a theory of equilibrium binding of symmetric bivalent haptens to cell surface antibody in the presence or absence of monovalent hapten. Bivalent haptens can link together antibodies to form linear chains or rings on cell surfaces. We show how to calculate the amount of any complex of bound bivalent hapten, monovalene fraction of antibody involved in complexes made up of two or more antibodies, i.e., the fraction of antibody that is cross-linked (Xpoly). We treat the case when the antibody on the cell surface, which is specific for the hapten, is homogeneous. For this case we prove a number of general properties about Xpoly: 1) Xpoly approaches zero at both high and low bivalent hapten concentration. 2) Xpoly becomes a maximum when the bivalent hapten concentration equals Amax, where Amax = 1/H + B/2. H is twice the equilibrium constant for the binding of a single hapten site to a single antibody site and B is the monovalent hapten concentration. 3) a plot of Xpoly vs the log of the bivalent hapten concentration is symmetric about the maximum value of Xpoly. We use these and other properties of Xpoly in this paper to clarify the relationship between cross-link formation and histamine release.     

Antibody catalyzed hydrolysis of a quorum sensing signal found in Gram-negative bacteria

N-(3-Oxo-acyl) homoserine lactones are used by Gram-negative bacteria to signal the establishment of specific population densities and coordinate population-wide gene expression. Herein we report the antibody-catalyzed hydrolysis of N-(3-oxo-acyl) homoserine lactone (AHL) using a reactive immunization strategy with a squaric monoester monoamide hapten. Kinetic analysis of the most efficient antibody revealed a modest k(cat), with AHL hydrolysis competitively inhibited by original squaric monoester monoamide hapten. These studies suggest that antibody catalysis could provide a new avenue for blocking quorum sensing in bacteria.     

Metalloantibodies: mercury(II)-dependent acyl transferases.

A new approach for the elicitation of metal-dependent catalytic antibodies for ester hydrolysis is described. A coordinatively unsaturated mercury complex 1-(Hg), has been utilized as a hapten to elicit antibodies that incorporate mercury(II) as a Lewis acid cofactor. From a panel of monoclonal antibodies generated to 1-(Hg), antibody 38G2 was found to hydrolyze the ester 3 in the presence of HgCl(2) [K(m)app(3)=345 microM; K(m)app(Hg(2+))=87 microM; k(cat)app/k(uncat)=3 x 10(2)]. This is the first example of a biocatalyst that enlists mercuric ion as a cofactor and it is anticipated that this approach will open new avenues for exploitation of metals thought previously beyond the scope of protein catalysts.     

Augmenting the efficacy of anti-cocaine catalytic antibodies through chimeric hapten design and combinatorial vaccination

Given the need for further improvements in anti-cocaine vaccination strategies, a chimeric hapten (GNET) was developed that combines chemically-stable structural features from steady-state haptens with the hydrolytic functionality present in transition-state mimetic haptens. Additionally, as a further investigation into the generation of an improved bifunctional antibody pool, sequential vaccination with steady-state and transition-state mimetic haptens was undertaken. While GNET induced the formation of catalytically-active antibodies, it did not improve overall behavioral efficacy. In contrast, the resulting pool of antibodies from GNE/GNT co-administration demonstrated intermediate efficacy as compared to antibodies developed from either hapten alone. Overall, improved antibody catalytic efficiency appears necessary to achieve the synergistic benefits of combining cocaine hydrolysis with peripheral sequestration.     

Hydrolysis of the 5'-p-nitrophenyl ester of TMP by the proofreading exonuclease (epsilon) subunit of Escherichia coli DNA polymerase III

The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (alpha, epsilon, and theta). The epsilon subunit is the 3'-5' proofreading exonuclease that associates with the polymerase (alpha) through its C-terminal region and theta through a 185-residue N-terminal domain (epsilon 186). A spectrophotometric assay for measurement of epsilon activity is described. Proteins epsilon and epsilon 186 and the epsilon 186.theta complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of k(cat) and K(M), confirming that the N-terminal domain of epsilon bears the exonuclease active site, and showing that association with theta has little direct effect on the chemistry occurring at the active site of epsilon. On the other hand, formation of the complex with theta stabilized epsilon 186 by approximately 14 degrees C against thermal inactivation. For epsilon 186, k(cat) = 293 min(-)(1) and K(M) = 1.08 mM at pH 8.00 and 25 degrees C, with a Mn(2+) concentration of 1 mM. Hydrolysis of pNP-TMP by epsilon 186 depended absolutely on divalent metal ions, and was inhibited by the product TMP. Dependencies on Mn(2+) and Mg(2+) concentrations were examined, giving a K(Mn) of 0.31 mM and a k(cat) of 334 min(-1) for Mn(2+) and a K(Mg) of 6.9 mM and a k(cat) of 19.9 min(-1) for Mg(2+). Inhibition by TMP was formally competitive [K(i) = 4.3 microM (with a Mn(2+) concentration of 1 mM)]. The pH dependence of pNP-TMP hydrolysis by epsilon 186, in the pH range of 6.5-9.0, was found to be simple. K(M) was essentially invariant between pH 6.5 and 8.5, while k(cat) depended on titration of a single group with a pK(a) of 7.7, approaching limiting values of 50 min(-1) at pH <6.5 and 400 min(-1) at pH >9.0. These data are used in conjunction with crystal structures of the complex of epsilon 186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by epsilon at high and low pH values.     

Enantioselective hydrolysis of naproxen ethyl ester catalyzed by monoclonal antibodies

This report described that a hapten of racemic phosphonate 3 designed as the mimic of the transition state of hydrolysis of naproxen ethyl ester was successfully synthesized from easily available 2-acetyl-6-methoxy-naphthalene 5. Then BALB/C mice were immunized and one of the monoclonal catalytic antibodies, N116-27, which enantioselectively accelerated the hydrolysis of the R-(-)-naproxen ethyl ester was given. The Michaelis-Menton parameter for the catalyzed reaction was K(M)=6.67 mM and k(cat)/k(uncat)=5.8 x 10(4). This enantioselective result was explained by the fact that the R-isomer of rac-hapten was more immunogenic than the S-isomer.     

Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.     

Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100

The beta-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl beta-D-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl beta-xylopyranosides substrates, in which leaving group pK(a)s were in the range of 3.96-10.32, revealed monotonic function of log(k(cat)) and no correlation of log(k(cat)/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of k(cat)/Km indicated two ionizable groups in the beta-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and k(cat), of p-nitrophenyl beta-D-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside. Increase of p-nitrophenyl-beta-D-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in k(cat)(app)=81 s(-1). Addition of alpha-methyl D-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-beta-D-xylopyranoside (50 mM) caused an acceleration of the beta-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, alpha-methyl D-xylopyranosyl-1,4-beta-D-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.     

Distances of tyrosine residues from a spin-label hapten in the combining site of a specific monoclonal antibody

The nuclear magnetic resonance spectra of an Fab fragment of a monoclonal antibody specifically directed against a nitroxide spin-label hapten have been recorded at different concentrations of the hapten. The hybridoma producing this antibody was grown on deuterated phenylalanine, tryptophan, and 3,5-dideuteriotyrosine or 2,6-dideuteriotyrosine. Difference spectra--without hapten minus with hapten--were calculated for each concentration of hapten. The difference spectra reveal five well-resolved singlet proton resonance signals from tyrosine deuterated in the 3,5-positions (H 2,6 Tyr) and nine from tyrosine deuterated in the 2,6-positions (H 3,5 Tyr). The measured intensities of these signals as a function of combining site occupation have been interpreted in terms of a theory involving intrinsic line widths (T2), the hapten off-rate (k), and distances to the paramagnetic center. Good agreement with theory is found for all of the isolated proton signals. The best estimate of k is 350 s-1; distances in the range 13 to less than 9 A are calculated. Extension of this analysis to other amino acids is discussed.     

Hapten synthesis for enzyme-linked immunoassay of the insecticide triazophos

Two haptens of the insecticide triazophos (O,O-diethyl O-[1-phenyl-1H-1,2,4-triazol-3-yl] phosphorothioate) were synthesized by introducing appropriate spacers in the O-ethyl site of the analyte molecular structure. First, thiophosphoryl chloride (PSCl(3)) reacts with methanol at low temperature to give O-ethyl dichlorothiophosphate. After reacting with 1-phenyl-3H-1,2,4-triazol, the O-ethyl dichlorothiophosphate was transformed into the intermediate O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate. Then the intermediate reacts with 4-aminobutyric acid and 6-aminobutyric acid to produce hapten I and hapten II, respectively. The molecule structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrum and mass spectrum. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was also developed to evaluate the two haptens. Results showed that the monoclonal antibodies with high titers were obtained after immunizing with protein conjugates of these haptens and that the immunoassay has high affinity and specificity to triazophos. These results suggested that the haptens were synthesized successfully and could be used for immunoassay for the rapid screening and sensitive determination of this insecticide.     

Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.     

The effect of pH and various cations on the GTP hydrolysis of rice heterotrimeric G-protein alpha subunit expressed in Escherichia coli

Previously, we reported the biochemical properties of RGA1 that is expressed in Escherichia coli (Seo et al., 1997). The activities of RGA1 that hydrolyzes and binds guanine nucleotide were dependent on the MgCl(2) concentration. The steady state rate constant (k(cat) ) for GTP hydrolysis of RGA1 at 2 mM MgCl(2) was 0.0075 +/- 0.0001 min(-1). Here, we examined the effects of pH and cations on the GTPase activity. The optimum pH at 2 mM MgCl(2) was approximately 6.0; whereas, the pH at 2 mM NH(4)Cl was approximately 4.0. The result from the cation dependence on the GTPase (guanosine 5'-triphosphatase) activity of RGA1 under the same condition showed that the GTP hydrolysis rate (k(cat)= 0.0353 min(-1)) under the condition of 2 mM NH(4)Cl at pH 4.0 was the highest. It corresponded to about 3.24-fold of the k(cat) value of 0.0109 min(-1) in the presence of 2 mM MgCl(2) at pH 6.0.     

Structural characterization and reversal of the natural organophosphate resistance of a D-type esterase, Saccharomyces cerevisiae S-formylglutathione hydrolase

Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 A resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme "acyl pocket". The W197I substitution enhances ySFGH reactivity with paraoxon by >1000-fold ( k i (W197I) = 16 +/- 2 mM (-1) h (-1)), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 A); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic acid may play a role in the regulation of a "D-type" esterase. The structure of the W197I variant is the first reported cysteine sulfenic acid in a serine esterase. We constructed five Cys60/W197I variants and show that introducing a positive charge near the oxyanion hole, W197I/C60R or W197I/C60K, results in a further enhancement of the rates of phosphorylation with paraoxon ( k i = 42 or 80 mM (-1) h (-1), respectively) but does not affect the dephosphorylation of the enzyme. We also characterized three histidine substitutions near the oxyanion hole, G57H, L58H, and M162H, which significantly decrease esterase activity.     

Analysis of active-site amino-acid residues of human serum paraoxonase using competitive substrates

Serum paraoxonase (PON1) is a calcium-dependent six-fold beta-propeller protein structurally similar to the di-isopropylfluorophosphatase (DFPase) found in the squid Loligo vulgaris. Human serum paraoxonase (HuPON1) has been shown to hydrolyze an array of substrates even though relatively little is known about its physiological role(s) or its catalytic mechanism. Through site-directed mutagenesis studies, designed from a DFPase-like homology model, and from a crystal structure of a hybrid PON1 molecule, amino-acid residues essential for enzyme function, including H115 and F222, have been identified. It was shown previously that, when H115 is replaced with tryptophan, the resulting enzyme hydrolyzes paraoxon but not phenyl acetate. This study shows that, when present simultaneously, phenyl acetate competitively inhibits paraoxon hydrolysis by H115W. Conversely, when F222 is replaced with tyrosine, mutant F222Y can hydrolyze phenyl acetate but not paraoxon. The presence of DFP, an inhibitor of both arylesterase and paraoxonase activities of wild-type HuPON1 (mean Ki=0.48+/-0.15 mM), has no effect on the ability of F222Y to catalyze the hydrolysis of phenyl acetate, suggesting that the F222Y mutant is unable to bind DFP. Together, the results suggest that, in wild-type HuPON1, H115 and F222 are important in determining substrate binding and specificity, but are not likely to be directly involved in substrate hydrolysis.     

Novel reactions catalysed by antibodies

New structural data on nonhydrolytic antibody catalysts gained over the past two years confirm that antibodies elicited against transition-state analogues function by differential stabilisation of the transition-state over the ground state through electrostatic, van der Waals, cation-pi and hydrogen-bonding interactions. The lack of chemical catalysis correlates with the low catalytic efficiency. Novel strategies that precisely position a key functional residue in the antibody catalyst combining site have therefore emerged, as demonstrated by crystallographic studies. Whereas antibodies with a bulky residue at position H100c of hypervariable loop H3 adopt different cavity shapes, other antibodies share a common deep combining site. This structural restriction might reflect the use of similar hydrophobic haptens to generate the antibody; novel hapten design or new immunisation strategies may, in the future, lead to more structurally diversified active sites.     

Structural and dynamical aspects of membrane immunochemistry using model membranes.

Three different phospholipid haptens have been synthesized, in which the haptenic group is the paramagnetic nitroxide (spin-label) group. These lipid haptens differ from one another in the length and composition of the molecular chain linking the 2,2,6,6-tetramethylpiperidinyl-N-oxy moiety to the phosphodiester group of the lipid. These lipid haptens have been incorporated at low molar concentrations (0.01 to 0.5 mol %) in liposomes containing various proportions of cholesterol and dipalmitoylphosphatidylcholine (DPPC). A study has been made of specific antinitroxide IgG (and Fab) binding to these liposomes, and the fixation of complement. From these studies we conclude: (a) For lipid haptens whose possible extension above the bilayer plane is limited (e.g., approximately 10-20 A), antibody binding and complement fixation depend strongly on the hapten structure and host lipid composition, because of steric limitations on the accessibility of lipid haptens to the binding sites in the protein. (b) Complement fixation by specific IgG antibodies directed against the nitroxide group as part of a lipid hapten depends strongly on the lateral mobility of the lipid hapten when its molar concentration in the plane of the membrane is of the order of 0.1 mol % or less. It is likely that this conclusion applies to many lipid haptens, and possibly other membrane components. (c) The inclusion of cholesterol in lipid membranes has at least two distinct effects on complement fixation involving lipid haptens. Through a steric effect on bilayer structure (probably involving lateral molecular ordering) cholesterol in phosphatidylcholine bilayers can enhance hapten exposure to antibody binding sites, enhance antibody binding, and thereby enhance complement fixation. It is likely that cholesterol also affects complement fixation at low hapten concentrations through a modification of membrane fluidity.     

Catalytic antibodies: hapten design strategies and screening methods

Catalytic antibodies have emerged as powerful tools for the efficient and specific catalysis of a wide range of chemical transformations. Generating antibody catalysts that achieve enzymatic efficiency remains a challenging task, which has long been the source of great interest both in the design of more effective haptens for immunization and in the development of more direct and efficient screening methods for the selection of antibodies with desired catalytic capacities. In this review, we describe the development of different hapten design strategies, including a transition state analog (TSA) approach, 'bait-and-switch' catalysis, and reactive immunization. We also comment on recent developments in the screening process that allow for a more efficient identification of antibody catalysts.     

Differential hydrolysis of homocysteine thiolactone by purified human serum (192)Q and (192)R PON1 isoenzymes

Human serum paraoxonase 1 (PON1) is a HDL-associated enzyme that catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates. Recently it has been suggested that a physiological substrate of serum PON1 is homocysteine thiolactone which is a putative risk factor in atherosclerosis. In this study, human (192)Q and (192)R PON1 isoenzymes were purified from the respective phenotype human serum, using a protocol consisting of ammonium sulfate precipitation and four chromatography steps: gel filtration, ion-exchange, non-specific affinity, and a second ion-exchange. Using paraoxon as substrate, overall purification fold was found as 742 for (192)R PON1 and 590 for (192)Q PON1. The final purified enzymes were shown as single protein bands close to 45kDa on SDS-PAGE and confirmed by Western blot. Substrate kinetics were studied with phenyl acetate, paraoxon and homocysteine thiolactone. Both PON1 isoenzymes showed mixed type inhibition with phenyl acetate. K(m) values of (192)Q and (192)R PON1 for homocysteine thiolactone were 23.5mM and 22.6mM respectively. For (192)R PON1, the V(max) was 2.5-fold and k(cat)/K(m) was 2.6-fold higher than those for (192)Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining (192)Q and (192)R PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment.     

Evidence that the mechanism of antibody-catalysed hydrolysis of arylcarbamates can be determined by the structure of the immunogen used to elicit the catalytic antibody

A kinetically homogeneous anti-phosphate catalytic antibody preparation was shown to catalyse the hydrolysis of a series of O-aryl N-methyl carbamates containing various substituents in the 4-position of the O-phenyl group. The specific nature of the antibody catalysis was demonstrated by the adherence of these reactions to the Michaelis-Menten equation, the complete inhibition by a hapten analogue, and the failure of the antibody to catalyse the hydrolysis of the 2-nitrophenyl analogue of the 4-nitrophenylcarbamate substrate. Hammett sigma-rho analysis suggests that both the non-catalysed and antibody-catalysed reactions proceed by mechanisms in which development of the aryloxyanion of the leaving group is well advanced in the transition state of the rate-determining step. This is probably the ElcB (elimination-addition) mechanism for the non-catalysed reaction, but for the antibody-catalysed reaction might be either ElcB or B(Ac)2 (addition-elimination), in which the elimination of the aryloxy group from the tetrahedral intermediate has become rate-determining. This result provides evidence of the dominance of recognition of phenolate ion character in the phosphate hapten in the elicitation process, and is discussed in connection with data from the literature that suggest a B(Ac)2 mechanism, with rate-determining formation of the tetrahedral intermediate for the hydrolysis of carbamate substrates catalysed by an antibody elicited by a phosphonamidate hapten in which phenolate anion character is minimized. The present paper contributes to the growing awareness that small differences in the structure of haptens can produce large differences in catalytic characteristics.