Peptide substrates for chymosin (rennin). Interaction sites in kappa-casein-related sequences located outside the (103-108)-hexapeptide region that fits into the enzyme's active-site cleft.
The role of individual amino acid residues in the 98-102 and 111-112 regions of bovine kappa-casein in its interaction with the milk-clotting enzyme chymosin (rennin) was investigated. to this end the tryptic 98-112 fragment of kappa-casein was modified in its N- and/or C-terminal part by chemical (guanidation, ethoxyformylation, repeated Edman degradation) and enzymic (carboxypeptidase) treatments. Further, use was made of short synthetic kappa-casein analogues in which His-102 had been replaced by Pro or Lys. All peptides and their derivatives were tested comparatively at various pH values for their ability to act as chymosin substrates via specific cleavage of the peptide bond at position 105-106. The results indicate that in the alternating 98-102 sequence (His-Pro-His-Pro-His) the His as well as the Pro residues contribute to the substrate activity with no predominant role of any one of these groups. Another interaction site is formed by the Lys residue at position 111 of the substrate. A model of the enzyme-substrate complex is proposed. Herein the 103-108 fragment of the substrate, to be accommodated within the enzyme's active-site cleft, is brought into position by electrostatic binding (via His-98, His-100, His-102 and Lys-111) near the entrance of the cleft. These interactions are strongly supported by Pro residues at positions 99, 101, 109 and 110 of the substrate, which act as stabilizers of the proper conformation of the substrate in the enzyme-substrate complex.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002364:Caseins; D012085:Chymosin / Q000378:metabolism*; D006639:Histidine / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000596:Amino Acids; D002364:Caseins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D006639:Histidine; D012085:Chymosin