Structure of the o-Aminocarboxylic Acid-binding Sites of Human Plasminogen ARGININE 70 AND ASPARTIC ACID 56 ARE ESSENTIAL FOR BINDING OF LIGAND BY KRINGLE

Kringle 4, an elastase fragment of plasminogen which carries one of the homologous o-aminocarboxylic acidbinding sites of the protein, has been subjected to chemical modification to identify the residues essential for ligand binding. Modification of kringle 4 with 1,2-cyclohexanedione or l-ethyl-3-(3-dimethylaminopropyl)carbodiimide has been found to abolish ligand binding as detected by the loss of the fragment’s affinity for lysine-Sepharose. Our results show that modification of a guanidino and a carboxyl group is responsible for loss of affinity. We suggest that the essential residues provide the positive and negative charges necessary for electrostatic binding of the ligand’s carboxylate and ammonium groups. In agreement with the proposed involvement of the essential residues in ligand binding, presence of ligand (e-aminohexanoic acid) in the reaction mixture prevents the reaction leading to affinity loss in the case of both reagents. Identification of the essential residues has been performed on tryptic peptides obtained from modified kringle 4 preparations. The results show that the fragment’s affinity for lysine-Sepharose is lost as a result of reaction of 1,2-cyclohexanedione with Arg-70 or reaction of the carbodiimide with Asp-56. The role assigned to Arg-70 and Asp-56 in kringle 4 probably is also valid in the case of the o-aminocarboxylic acid-binding site of kringle 1 since both essential residues are preserved in homologous positions of this structure. Conversely, both essential charged residues are replaced by neutral amino acids in kringle 5, explaining why this structure is devoid of an w-aminocarboxylic acid-binding site.