Substrate Recognition and Saturation Kinetics in de Novo Designed Histidine-Based Four-Helix Bundle Catalysts

Abstract

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 are 0.075 M-1 s-1 and 0.135 M-1 s-1. MNKR catalyzed hydrolysis of the fumarate follows saturation kinet...