Catalysis of the Kemp elimination by antibodies elicited against a cationic hapten

Genre-Grandpierre, Arnaud; Tellier, Charles; Loirat, M.J.; Blanchard, Dominique; Hodgson, David R. W.; Hollfelder, Florian; Kirby, Anthony J.

doi:10.1016/s0960-894x(97)10003-8

Cited by 31 publications

(41 citation statements)

References 21 publications

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“…A frequently used comparison is the ratio of the value of k cat ∕K M for AlleyCat versus the second order rate constant for acetate in water, which amounts to 2.8 × 10 5 in this case. The value of k cat ∕K M for AlleyCat (5.8 AE 0.3 M −1 s −1 ) is within the same range as observed for catalytic antibodies (8,28,29) …”

Section: Discussionsupporting

confidence: 76%

Design of a switchable eliminase

Korendovych

Kulp

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

120

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The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.protein design | catalysis | pKa T he design of enzyme-like catalytic proteins is a challenging endeavour that has been approached using combinatorial (1) and computational strategies (2), as well as by following simple chemical principles (3, 4). The Kemp elimination (5) (Scheme 1) is an extensively studied benchmark for catalyst design (6-8) as a model for enzymatic C-H bond abstraction. Previously, computational methods were employed to alter the active sites of natural enzymes to catalyze this reaction (7). Mutation of a dozen or more side chains resulted in catalysts that showed rate enhancements of 10 4 to 10 5 relative to a given reference rate. Here, by combining a few physical principles with modern computational tools, we show that a single mutation can confer similar catalytic efficiency into a much simpler and allosterically switchable scaffold that previously had no catalytic activity. This approach circumvents often contentious questions concerning residual activities of the enzymatic framework or the appropriate reference background rate for expression of a "rate enhancement" while also suggesting a practical approach to the design of catalytically amplified sensors.Here we applied a minimalist approach to protein design (9-11), which emphasizes the use of the simple scaffolds and the min...

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Section: Discussionsupporting

confidence: 76%

Design of a switchable eliminase

Korendovych

Kulp

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

120

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“…20). Both reactions would be catalyzed by a residue acting as a general base to abstract the ␣-proton of ␤-␥ unsaturated ketone in allylic isomerization or the proton of the isoxazole ring in the Kemp elimination.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, antibodies resulting from these strategies have been found to catalyze different reactions that use the same mechanism, provided that the various substrates possess the common chemical function with which the induced residue can react (10,36,37). This finding is also illustrated by antibody 4B2, which catalyzes both the Kemp elimination (20) and the allylic rearrangement of a ␤-␥ unsaturated ketone.…”

Section: Discussionmentioning

confidence: 98%

Structural evidence for a programmed general base in the active site of a catalytic antibody

Golinelli‐Pimpaneau

Gonçalves

Dintinger

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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The crystal structure of the complex of a catalytic antibody with its cationic hapten at 1.9-Å resolution demonstrates that the hapten amidinium group is stabilized through an ionic pair interaction with the carboxylate of a combining-site residue. The location of this carboxylate allows it to act as a general base in an allylic rearrangement. When compared with structures of other antibody complexes in which the positive moiety of the hapten is stabilized mostly by cation-interactions, this structure shows that the amidinium moiety is a useful candidate to elicit a carboxylate in an antibody combining site at a predetermined location with respect to the hapten. More generally, this structure highlights the advantage of a bidentate hapten for the programmed positioning of a chemically reactive residue in an antibody through charge complementarity to the hapten.A llylic rearrangements play a fundamental role in the biosynthesis of terpenes and steroid hormones and in the biodegradation of fatty acids (1). The enzymatic rearrangement of ␤-␥ unsaturated ketones requires a general base, usually a carboxylate, to abstract the ␣-proton of the ketone ( Fig. 1; refs. 1 and 2). This reaction leads to a dienol or dienolate high-energy intermediate 3 that does not possess a charge complementing that of the carboxylate. Therefore, antibodies elicited against a hapten that mimics the transition state or the dienol intermediate of allylic rearrangement would acquire a properly positioned general base carboxylate to catalyze this reaction only by serendipity. However, the extensive experience available on catalytic antibodies (3, 4) and the structures of catalytic antibodies elicited by transition state analogue haptens show that properly positioned chemically reactive residues are rarely present in the active site (5). An alternative approach that aims to generate functional residues, also termed ''bait and switch'' (6, 7), uses a charged hapten to induce the required complementary charged residue (8, 9). Haptens containing a positive charge have provided antibody catalysts for important reactions such as acyl transfer (7), elimination (9, 10), and phosphodiester hydrolysis (11). However, in the absence of structural data, it is not possible to establish unambiguously the nature and identity of the catalytic residue that has been induced and the relationship between the location of the haptenic charge and the position of the catalytic residue in the antibody combining site.Indeed, in the few cases in which the use of a hapten containing a positively charged moiety successfully induced catalytic antibodies and in which the structure of the hapten-antibody complex was determined, there was no negatively charged residue in the active site directly facing the positive charge, but stabilization of the haptenic charge was mediated mostly by cation-interactions (12-14). Herein, we report the structure, at 1.87-Å resolution, of the complex of an antibody catalyzing an allylic rearrangement with its cationic hapten. We provide direct ...

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“…To ensure that the two Fv domains in the MoaFv were fully active, we carried out an active site titration by following the inhibition of the antibody-catalyzed Kemp reaction upon addition of the 2-(4-aminobenzylamino)-3,4,5,6-tetrahydropyridinium hapten. 25 (Figure 4), demonstrating that the MoaFv is fully active as a bivalent pseudo-F(ab 0 ) 2 . The fact that the stoichiometry was not exactly 2 is probably due to the partial inactivation of the MoaFv during the time course of the experiment.…”

Section: Oligomerization State Of Moafv and Bivalencymentioning

confidence: 96%