Catalysis of Hydrolysis and Transesterification Reactions of p-Nitrophenyl Esters by a Designed Helix−Loop−Helix Dimer

Abstract

KO-42, a polypeptide with 42 amino acid residues has been designed to fold into a hairpin helix−loop−helix motif that dimerizes and forms a four-helix bundle. The solution structure of the folded KO-42 dimer has been determined by NMR and CD spectroscopy and ultracentrifugation. On the surface of the folded polypeptide a reactive site has been engineered that is capable of catalyzing acyl-transfer reactions of reactive esters. The reactive site of KO-42 contains six histidine residues with perturbed pK a values. The pK as of His-15, His-30, and His-34 are close to 5, whereas those of His-11, His-19, and His-26 are close to 7, with nonideal titration curves. The second-order rate constant for the KO-42 catalyzed hydrolysis of mono-p-nitrophenyl fumarate at pH 4.1 and 290 K is 0.1 M-1 s-1, which is 1140 times larger than that of the 4-methylimidazole (4-MeIm) catalyzed reaction, 8.8 × 10-5 M-1 s-1. The second-order rate constant for the KO-42 catalyzed transesterification of mono-p-nitrophenyl fumarate to form the corresponding trifluoroethyl ester in 10 vol % trifluoroethanol at pH 4.1 and 290 K is 0.052 M-1 s-1 which is 620 times larger than that of the 4-MeIm catalyzed reaction, 8.4 × 10-5 M-1 s-1. KO-42 catalyzes the corresponding reactions of other p-nitrophenyl esters with similar rate enhancements. At pH 4.1 in aqueous solution where the rate constant ratio k 2(KO-42)/k 2(4-MeIm) is larger than 103 the predominant reactive species of KO-42 have unprotonated histidines flanked by protonated histidines. The kinetic solvent isotope effect at pH 4.7 is 2.0 which shows that isotopic fractionation occurs in the transition state. The kinetic solvent isotope effect at pH 6.1 is 1.1 which shows that there is neither general acid−general base catalysis nor strong hydrogen bonding in the transition state of the rate-limiting reaction step at that pH. The results suggest that at low pH the dominant catalytic species functions through a mechanism where unprotonated nucleophilic histidines are flanked by protonated histidines that bind to one or both of the ester oxygens in the transition state.

Installing hydrolytic activity into a completely de novo protein framework

“…23 The overall catalytic efficiency (kcat/KM = 3.7 ± 0.6 M -1 s -1 ) is approximately equal to previous redesign attempts (Table 2 entries 3, 8&9). In our case, the reaction is turned over expelling acetate, and it is not driven by other nucleophilic side chains near the active site, which can become irreversibly acylated as the reaction proceeds contributing to observed kinetic profiles.…”

“…[20][21][22] Although more challenging, this would carry advantages over redesign approaches: firstly, the designer could select and control all, or at least many of the residues, and so engineer the complete construct predictably; secondly, the resulting proteins could be made to function under conditions away from those required by natural proteins; and finally, success in this area would provide the acid test of our understanding of enzyme structure and function. Towards this fully de novo effort, successful designs of hydrolases have included: the decoration of small protein-folding motifs with His residues; 23,24 the employment of Zn 2+ cations as Lewisacidic cofactors; [25][26][27][28] and the identification of catalytically active proteins in combinatorial libraries of sequences patterned to form to all- or all- protein folds. 29 One of the most productive areas in the de novo design of protein structure has been for coiled-coil assemblies.…”

Enzyme-like proteins by computational design

“…The second-order rates for these peptides were determined under different conditions than those used here, making direct comparisons difficult. At pH 5.1, rates of 0.29 M Ϫ1 sec Ϫ1 and 0.056 M Ϫ1 sec Ϫ1 have been reported (39,40). Extrapolation based on the pH rate profiles (39) reported for the hydrolysis of similarly activated esters suggests rates of Ϸ0.7 and 0.2 M Ϫ1 sec Ϫ1 for PNPA hydrolysis at pH 7, respectively.…”

Design of a switchable eliminase

“…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)(7)(8) as a model for enzymatic C-H bond abstraction.…”