A highly enantioselective aminopeptidase from sunflower seed—Kinetic studies, substrate mapping and application to biocatalytic transformations

The sunflower seed (Helianthus annuus L.) major aminopeptidase primary specificity has been assessed with a number of specially designed amino acid amides and alkyl esters. Studies with amino acid 4-nitroanilides with straight alkyl side chains suggest the enzyme possesses an S1 hydrophobic binding site of limited size and effects of substrate sorption are present in both its binding and turnover. The enzyme S1′-subsite is able to accommodate a number of different leaving groups—primary amide, one- and two-ring condensed aromatic structures, amino acid residues and alkyl ester moieties. The hydrolysis of amide substrates is affected by both their P1 and the P1′ part. The replacement of the amide leaving group with an ester one tents to minimize the P1-part effect. The enzyme's high enantioselectivity towards the configuration of the substrate P1-part has been successfully utilized for resolution of racemic amino acid amides.