Polyclonal antibody-catalysed hydrolysis of a β-lactam

Ostler, Elizabeth L.; Resmini, Marina; Boucher, Guillaume; Romanov, Nickolas; Brocklehurst, Keith; Gallacher, Gerard

doi:10.1039/b110135h

Cited by 8 publications

(6 citation statements)

References 18 publications

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“…In the present work, the little-investigated anti-phosphate antibody preparation PCA 271-22 and the newly elicited anti-phosphonate antibody preparation PCA 2649-16 were investigated kinetically. Use of the two pairs of isomeric substrates (2a and 2b for PCA 271-22, and 6a and 6b for PCA 2649-16) provided strong evidence for the absence of enzyme contaminants, as has been found for other PCA preparations [2,18]. As is the case for all other PCA preparations investigated in these laboratories, catalysis by PCA 271-22 and PCA 2649-16 was found to obey single-site saturation kinetics (Figure 2).…”

Section: Catalytic Antibody Preparationssupporting

confidence: 60%

“…In contrast, two different carboxylesterases failed to discriminate substantially between the two substrates, as did whole serum from a non-immunized sheep or from a sheep immunized with an immunogen structurally unrelated to the substrate (a derivative of 3-O-methylnoradrenaline). This approach was also used more recently [18] to demonstrate that hydrolysis of the 4-nitrophenyl β-lactam (3a) catalysed by one of our anti-(4-nitrophenyl phosphate) IgG preparations is antibody-mediated, by showing that the 2-nitrophenyl β-lactam (3b) is not a substrate. The evidence provided by using isomeric substrates is complemented by the fact that catalysis by polyclonal antibody preparations of reactions for which there are no known enzymes has been demonstrated.…”

Section: Improvement In Hydrolytic Antibody Activity By Change In Hapmentioning

confidence: 94%

“…Both anxieties have been shown to be without foundation. Thus, as far as the first is concerned, catalysis by all of the polyclonal IgG preparations investigated to date in different laboratories has not deviated from single-site (Michaelis-Menten) saturation kinetics (e.g [2][3][4][10][11][12][13][14][15][16][17][18]). Given the heterogeneous nature of the polyclonal antisera, this observation may be regarded as counter-intuitive.…”

Section: Improvement In Hydrolytic Antibody Activity By Change In Hapmentioning

confidence: 99%

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Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the ‘flexibility’ hypothesis

Gul

Sonkaria

Pinitglang

et al. 2003

Biochemical Journal

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To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in k(cat)/k(non-cat) and a 100-fold advantage in the proficiency constant, k(cat)/k (non-cat) x K(m). Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.

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Section: Catalytic Antibody Preparationssupporting

confidence: 60%

Section: Improvement In Hydrolytic Antibody Activity By Change In Hapmentioning

confidence: 94%

Section: Improvement In Hydrolytic Antibody Activity By Change In Hapmentioning

confidence: 99%

See 1 more Smart Citation

Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the ‘flexibility’ hypothesis

Gul

Sonkaria

Pinitglang

et al. 2003

Biochemical Journal

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“…These include anti‐phosphonamidate antibody 43C97 and polyclonal antiserum PCA 270‐29,8 both of which catalyze the hydrolysis of activated p ‐nitroanilides; primary‐amide‐hydrolyzing antibodies 13D119 and BL25,10 elicited against a phosphinate and a boronate hapten, respectively; and an amidase antibody that requires an external nucleophilic cofactor 11 . β ‐Lactamase activity has been described in an antiserum designed for carbonate hydrolysis,12 and in antibodies obtained by the anti‐idiotype approach13 or by reactive immunization 14. The intense research effort in this area has also fostered significant progress in the design of transition‐state analogues10, 15 and in the development of new immunization and selection strategies 14, 16…”

Section: Introductionmentioning

confidence: 99%

A Catalytic Antibody Programmed for Torsional Activation of Amide Bond Hydrolysis

Aggarwal

Benedetti

Berti

et al. 2003

Chemistry A European J

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Amidase antibody 312d6, obtained against the sulfonamide hapten 4 a that mimics the transition state for hydrolysis of a distorted amide, accelerates the hydrolysis of the corresponding amides 1 a-3 a by a factor of 10(3) at pH 8. The mechanisms of both the uncatalyzed and antibody-catalyzed reactions were studied. Between pH 8 and 12 the uncatalyzed hydrolysis of N-toluoylindoles 1 a and 3 a shows a simple first-order dependence on [OH(-)], while hydrolysis of 3 a is zeroth-order in [OH(-)] below pH 8. The pH profile for hydrolysis of the corresponding tryptophan amide 2 a is more complex due to the dissociation of the zwitterion into an anion with pK(a) 9.74; hydrolysis of the zwitterionic and the anionic form of 2 a both show simple first-order dependence on [OH(-)]. Absence of (18)O exchange between H(2) (18)O/(18)OH(-) and the substrate, a normal SKIE for both 1 a (k(H)/k(D)=1.12) and 3 a (k(H)/k(D)=1.24) and the value of the Hammett constant rho for hydrolysis of p-substituted amides 3 a-e are consistent with an ester-like mechanism in which formation of the tetrahedral intermediate is rate-determining and the amine departs as anion. The 312d6-catalyzed hydrolysis of 3 a was studied between pH 7.5 and 9, and its independence of pH in this range indicates that water is the reacting nucleophile. Hydrolysis of 3 a is only partially inhibited by the sulfonamide hapten, and this indicates that non-specific catalysis by the protein accompanies the specific process. Only the nonspecific process is observed in the hydrolysis of amides 3 with para substituents other than methyl. Binding studies on the corresponding series of p-substituted sulfonamides 5 a-e confirm the high specificity of antibody 312d6 for p-methyl substituted substrates.

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“…It is important to emphasize that (i) polyclonal IgG preparations investigated to-date in different laboratories have not deviated from single-phase (Michaelis-Menten) saturation kinetics (e.g. see references cited in [19]), (ii) polyclonal preparations are free from contamination by enzymes, as shown in our laboratories by the use of isomeric substrates [7,20] and in other laboratories by the observation of catalysis of reactions for which there are no known enzymes [14,21], and (iii) the generation of PCAs by immunization with a transition-state analogue [7,[22][23][24] is a good way to sample the entirety of the immune response to the haptenic moiety of an immunogen. Catalysis of the hydrolysis of the substrate 5e (4-CH 3 O) was too weak to be quantified reliably when using the concentration of PCA 271-100 available.…”

Section: Kinetic Characteristicsmentioning

confidence: 84%

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

Boucher

Said

Ostler

et al. 2007

Biochemical Journal

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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.

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Polyclonal antibody-catalysed hydrolysis of a β-lactam

Abstract: We report the first example of antibody-catalysed hydrolysis of a beta-lactam where the antibodies were generated by a simple transition-state analogue; in this example the antibodies are polyclonal.

Cited by 8 publications

References 18 publications

Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the ‘flexibility’ hypothesis

Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the ‘flexibility’ hypothesis

A Catalytic Antibody Programmed for Torsional Activation of Amide Bond Hydrolysis

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

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