Enzyme-substrate interactions in the hydrolysis of peptide substrates by thermitase, subtilisin BPN', and proteinase K.
Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.
D002918:Chymotrypsin / Q000494:pharmacology; D019286:Endopeptidase K; D010450:Endopeptidases / Q000494:pharmacology*; D006868:Hydrolysis; D007700:Kinetics; D010455:Peptides; D011487:Protein Conformation; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013381:Subtilisins / Q000494:pharmacology*
D010455:Peptides; D010450:Endopeptidases; D012697:Serine Endopeptidases; D013381:Subtilisins; C022728:thermitase; D002918:Chymotrypsin; D019286:Endopeptidase K