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.