SUBSTRATE RECOGNITION AND SATURATION KINETICS IN DE-NOVO DESIGNED HISTIDINE-BASED 4-HELIX BUNDLE CATALYSTS

Designed four-helix bundle proteins with reactive sites based on the c ooperativity of HisH(+)-His pairs in helical sequences catalyze acyl-t ransfer reactions of p-nitrophenyl esters with large rate enhancements . The function of the HisH(+)-His site has been expanded by the introd uction of flanking residues to provide recognition of substrate carbox ylate and hydrophobic residues. The second-order rate constants for th e MN 42 catalyzed hydrolysis of p-nitrophenyl acetate and of mono-p-ni trophenyl 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 0.02 7 M-1 s(-1), respectively. The reactive site of MN-42 contains only hi stidine residues. The sequence of MNKR is the same as that of MN-42 ex cept that one Lys and one Arg residue have been introduced in the adja cent helix to flank the HisH(+)-His site and the resulting second-orde r rate constants an 0.075 M-1 s(-1) and 0.135 M-1 s(-1). MNKR catalyze d 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 secon d-order rate constants for the JNIII catalyzed hydrolysis of p-nitroph enyl acetate and of p-nitrophenyl valerate are 0.007 M-1 s(-1) and 0.0 97 M-1 s(-1), respectively, and binding of the aliphatic group increas es the rate constant by more than one order of magnitude. Chiral recog nition by de novo designed polypeptides has been demonstrated for the first time, and the hydrolysis of the p-nitrophenyl ester of D-norleuc ine has been catalyzed with a second-order rate constant that is twice as large as that of the L-norleucine ester.