Substrate recognition and saturation kinetics in de novo designed histidine-based four-helix bundle catalysts

Designed four-helix bundle proteins with reactive sites based on the cooperativity of HisH(+)-His pairs in helical sequences catalyze acyl-transfer reactions of p-nitrophenyl esters with large rate enhancements. The function of the HisH(+)-His site has been expanded by the introduction of flanking residues to provide recognition of substrate carboxylate and hydrophobic residues. The second-order rate constants for the MN 42 catalyzed hydrolysis of p-nitrophenyl acetate and of mono-p-nitrophenyl fumarate, under conditions of excess catalyst over substrate, in aqueous solution at pH 5.1 and 290 K are 0.030 M-1 s(-1) and 0.027 M-1 s(-1), respectively. The reactive site of MN-42 contains only histidine residues. The sequence of MNKR is the same as that of MN-42 except that one Lys and one Arg residue have been introduced in the adjacent helix to flank the HisH(+)-His site and the resulting second-order rate constants an 0.075 M-1 s(-1) and 0.135 M-1 s(-1). MNKR catalyzed hydrolysis of the fumarate follows saturation kinetics with a k(cat)/K-M of 0.17 M-1 s(-1) which is 230 times larger than the second-order rate constant of the 4-methyl imidazole catalyzed reaction. The second-order rate constants for the JNIII catalyzed hydrolysis of p-nitrophenyl acetate and of p-nitrophenyl valerate are 0.007 M-1 s(-1) and 0.097 M-1 s(-1), respectively, and binding of the aliphatic group increases the rate constant by more than one order of magnitude. Chiral recognition by de novo designed polypeptides has been demonstrated for the first time, and the hydrolysis of the p-nitrophenyl ester of D-norleucine has been catalyzed with a second-order rate constant that is twice as large as that of the L-norleucine ester.