POLYPEPTIDES WITH SUPERSECONDARY STRUCTURES AS TEMPLATES IN RATIONAL CATALYST DESIGN - CATALYSIS OF SELF FUNCTIONALIZATION BY DESIGNED HELIX-LOOP-HELIX MOTIFS

Three designed polypeptides with 42 amino acids each, SA-42, RA-42 and PA-42, were engineered to catalyse acyl-transfer reactions of mono-p- nitrophenylfumarate I. The second-order rate constants of the peptides for the formation of p-nitrophenol were larger than that of the backg round reaction with factors of 331, 1750 and 1937, respectively. The b ackground reaction is the reaction between I and trifluoroethoxide ion to form the trifluoroethyl ester, The second-order rate constant for the 4-methylimidazole catalysed reaction between I and trifluoroethoxi de ion was 211 times larger than that of the background reaction and t he similarity in rate constants suggests that the histidine residues i n the polypeptides act as nucleophilic catalysts, The observed rate in creases in RA-42 and PA-42 over that of SA-42 identifies the location of the reaction centre, The identity of the reaction products in the R A-42 catalysed reaction was established by NMR spectroscopy and mass s pectrometry, The side chain of ornithine-15 was acylated by the fumary l residue, Thus, in a fast and selective second reaction step the fuma ryl group was transferred from histidine-11 to the side chain of ornit hine-15. The ability of a designed helix-loop-helix motif, RA-42, to c atalyse its own functionalization is thereby demonstrated for the firs t time.