COFACTOR-INDUCED REFINEMENT OF CATALYTIC ANTIBODY ACTIVITY : A METAL-SPECIFIC ALLOSTERIC EFFECT

We recently described the development of the strategy of reactive immunization for the production of two catalytic antibodies for the aldol reaction.1 These catalysts accept a tremendous variety of substrates in aldol addition, aldol condensation, and retro-aldol reactions.2 They also catalyze a mechanistically related decarboxylation of â-keto acids.3 A striking example of their effective promiscuity is the highly enantioselective intramolecular aldol condensation to give the Wieland-Miescher ketone, using a catalyst developed by immunization with a hapten only remotely similar to the reaction substrate.4 An X-ray crystal structure of one of these aldol catalysts (antibody 33F12) has revealed the presence of a single lysine side chain at the bottom of a large binding pocket lined with hydrophobic residues.2a This is consistent with evidence that catalysis occurs via enamine intermediates,1,2 characteristic of class I aldolases.5 Along with the ability of metal complexes to assist in the biocatalysis of the aldol reaction, the presence of potential metal-binding groups from several tyrosine and tryptophan residues in the putative antibody active site prompted us to screen the reactivities of these systems with a variety of added metal ions. We report here that “hard” metal ions typically utilized by class II aldolases do not perturb catalysis of the aldol reaction by our antibodies. In contrast, palladium(II) additives improve the rate and enantioselectivity of the process, but only for substrates closely resembling the hapten used for the generation of the antibody. The reaction of aldehyde 1 with acetone (eq 1)1 was used to screen the effect of added metal ion. Initial rates in the presence of the following species at 20, 50, and 150 μM were found to be indistinguishable ((5%) from the rates of the aldol reaction in the absence of additive under a standard set of conditions in the saturation regime of catalytic turnover (2 μM antibody;6 100 μM 1; 5% (v/v) acetone; phosphate/saline (PBS) buffer at pH 7.4; 22 ( 1 °C): LiCl, MgNO3, CaSO4, BaCO3, ZrOCl2, CrCl2, CrCl3, MnCl2, FeSO4, FeCl3, RuCl3, CoCl2, NiCl2, K2PtCl4, PtCl4, CuCl2, ZnCl2, CdCl2, HgCl2 CeCl3, LaCl3, La(OTf)3, Eu(OTf)3, YbCl3, and Yb(OTf)3. Visible precipitation of the antibodies, with concomitant reduction of the apparent reaction rate, often occurs at metal ion concentrations higher than 150 μM. In contrast, VOSO4 at relatively high concentration (100-300 μM relative to 2-5 μM antibody) was found to inhibit the aldol reactions of 1 and 2 with acetone in direct proportion to the amount of metal added,7 without antibody precipitation.8 Palladium(II) is the only metal species tested to have a significant effect on the antibody system at low concentrations. Thus, the standard aldol reaction (1 + acetone), catalyzed by both 38C2 and 33F12 (2 mM), is accelerated by a variety of PdII salts [K2PdCl4 and Na2PdCl4, Figure 1A; Pd(OAc2) and PdSO4, data not shown] and palladium amine complexes [Pd(en)Cl2 and transPd(NH3)2Cl2, Figure 1B,C]. The magnitude of the relative rate (krel) is roughly proportional to metal concentration below 20 mM, often remaining detectable with as little as a 2-fold excess of Pd with respect to the concentration of antibody active sites.10 Higher concentrations of PdII lead to apparent reaction inhibition or attenuation of the acceleratory effect, which is attributed to removal of active antibody from the reaction mixture by increased precipitation as metal concentrations are raised.11 Pd(en)Cl2 (en ) ethylenediamine) consistently induces much less observable precipitation than the other palladium species; accordingly, accelerated rates are observed at substantially higher concentrations of this complex than for other Pd sources. Pd-induced rate accelerations are also observed at higher antibody concentrations and in water instead of PBS buffer.7 Michaelis-Menten kinetic parameters measured for the 38C2catalyzed reaction reveal an improvement in kcat and a small increase in Km in the presence of PdII (Table 1). Control experiments show that Pd(en)Cl2-antibody binding is reversible12 (dissociation constant ≈ 1 μM)13 without causing functional