A new substrate and two inhibitors applicable for thermitase, subtilisin BPN' and α-chymotrypsin. Comparison of kinetic parameters with customary substrates and inhibitors

A new chromogenic substrate and two inhibitors with the common peptide sequence X-Ala-Ala-Phe-Y have been synthesized, and were found to be of higher efficiency as hitherto available customary substrates and inhibitors for thermitase, subtilisin BPN' and α-chymotrypsin. The proteolytic coefficient k(cat)/K(m) for the hydrolysis of the substrate Suc-Ala-Ala-Phe-pNA is about 80 times higher in the case of thermitase and subtilisin BPN' and about 20 times higher for α-chymotrypsin compared with Suc-Ala₃-pNA and Suc-Phe-pNA, respectively. The irreversible inhibitory effect of Z-Ala₂-Phe-CH₂Cl compared with Z-Phe-CH₂Cl is 280 times greater for thermitase and subtilisin BPN' and 50 times for α-chymotrypsin. The corresponding methyl ketone Z-Ala₂-Phe-CH₃ is a high affinity competitive inhibitor for thermitase but not for the two other enzymes.