It is known that the serine protease subtilisin pos-; sesses a specifity for longer peptide amides as oppo- sed to simpler amides [1,2]. Since ester substrates of the serine protease chymotrypsin generally are hydro- lyzed much more rapidly (10 s times) [3] than amides, the subtilisin-catalyzed hydrolyses of specific peptide esters should be much more rapid than those previous- ly studied. Two substrates used here have the highest known reaction rate (k2/Ks) with subtilisin. These rates, for methyl esters, approximate the rates of the best substrates of a-chymotrypsin [4] which are more highly activated p-nitrophenyl esters. Since in the most specific substrates, the value of k2 may exceed kl (eq. 1), the value ofkcat/K m may reduce to kl. [4] kl k2 E+S~ ES'~,I~EP2 E+ P2 (1) It is evident that the pH profile ofk~ need not be identical to that of k2/K s. [5] Therefore pH profiles of the various hydrolyses were obtained. Since litera- ture data [ 1,2] concerning the peptide amides similar to the esters is in the form of relative rate data, bin- ding constants of the amides to subtilisin were ob- tained. These were measured by observing inhibition of the second-order subtilisin-catalyzed hydrolysis of Z-Gly-OnP (see later).