THE SITE-SELECTIVE INCORPORATION OF A NAD(-LOOP-HELIX POLYPEPTIDE MOTIF() COFACTOR MIMIC INTO A FOLDED HELIX)

LA-42, a polypeptide with 42 amino acid residues, has been designed to fold into a hairpin helix-loop-helix motif that can dimerise in solut ion to form a four-helix bundle. On the surface of the folded motif a reactive site has been introduced that contains a histidine and a lysi ne residue in a helical sequence. The reaction between the His-Lys sit e and a p-nitrophenyl ester has previously been shown to lead to the s ite-selective formation of an amide at the side chain of the flanking lysine residue. An N-methylnicotinoyl group has now been incorporated into LA-42, in a reaction between the peptide and the N-methylnicotini c acid ester in aqueous solution at pH 5.9, to form a template for the engineering of selective catalysts for the reduction of carbonyl comp ounds. The formation of an amide bond between LA-42 and the cofactor m imic was established by electro-spray mass spectrometry and NMR spectr oscopy and the reduction of the N-methylnicotinoyl residue by sodium d ithionite in aqueous solution at pH 6.5-7 was demonstrated by UV and N MR spectroscopy. Key problems with NAD(+)/NADH models in aqueous solut ion include poor solubility of the ox and/or red forms of the catalyst s and short lifetimes of the red form due to hydrolysis of the enamine . Both the red and the ox forms of the peptide-linked nicotinoyl cofac tor are soluble in aqueous solution, which is a necessary condition fo r the development of turnover systems, and the lifetime of the reduced form of the polypeptide catalyst has been increased by more than a fa ctor of three over that of the 1-methylnicotinamide.