Molecular mechanism of enantioselective proton transfer to carbon in catalytic antibody 14D9

Abstract

Catalytic antibody 14D9 catalyzes the enantioselective protonation of prochiral enol ethers with high enantioselectivity (>99% ee) and a practical turnover (k(cat) = 0.4 s(-1)), allowing for preparative scale applications. This antibody represents one of the rare examples of catalytic antibodies promoting acid-catalyzed processes. Antibody 14D9 was cloned and expressed as a chimeric Fab fragment in Escherichia coli. Crystal structures of Fab 14D9 as apo form and of its close analog 19C9 in complex with the transition state analog were determined at 2.8-A resolution. A series of site-directed mutagenesis experiments was carried out to probe the role of individual active-site amino acids. Proton transfer to carbon is catalyzed by a hydrogen bond network formed by the side chains of Asp(H101) and Tyr(L36) with a water molecule serving as a relay. The intermediate oxocarbonium ion formed during the protonation step is trapped by the same water molecule, resulting in an overall syn-addition of water to the enol ether's double bond. The enantioselectivity is caused by steric crowding at the active site, mainly because of the side chain of Phe(H84). The 20-fold lower activity of 19C9 compared with 14D9 was traced down to residue Thr(L46), which forms a nonproductive hydrogen bond with the catalytic residue Asp(H101), which competes with the critical Asp(H101)-Tyr(L36) hydrogen bond and therefore reduces catalytic efficiency. The catalytic activity of 19C9 was restored to that of 14D9 by using either site-directed mutagenesis (Thr(L46)Ala) or chain shuffling.

Fig. 1.

Enantioselective protonation reaction catalyzed by antibody 14D9.

Fig. 2.

Stereoview of the binding pocket of antibody 19C9 with bound hapten. Hydrogen-bonding distances are denoted by black dashed lines. The red line indicates the distance (4.4 Å) between the catalytic Asp^H101 and the quaternary ammonium group of the hapten. Water W89 is shown as magenta spheres.

Fig. 3.

Fv domain gene sequence alignment of Ab 14D9 and 19C9. The identical residues are shown in red; different residues are shown in blue or black.

Fig. 4.

Stereoview of the 14D9 binding pocket with docked substrate. No energy minimization was performed.

Fig. 5.

Proposed mechanism of protonation reaction catalyzed by antibody 14D9. The rate-limiting step is a proton transfer from Asp^H101 to Re-face of enol ether by means of a water molecule. This water molecule then attacks the oxocarbonium intermediate 4 to form hemiketal 5 as the primary reaction product.

Fig. 6.

Stereoview of the 14D9 binding pocket with docked hemiketal 5.