N-anthraniloyl-Ala-Ala-Phe-4-nitroanilide, a highly sensitive substrate for subtilisins

A new substrate for subtilisins, anthraniloyl-Ala-Ala-Phe-4-nitroanilide, has been synthesized and characterized. The peptide is a fluorogenic substrate that is intramolecularly quenched without loss of its chromogenic properties and offers a possibility for double-assay kinetic analysis. The kinetic parameters determined for subtilisin Carlsberg are Km = 0.004 mM, kcat = 104 s-1, and those for subtilisin BPN' are Km = 0.020 mM, kcat = 49 s-1. The substrate is extremely sensitive for subtilisins; the specificity constants are 10-fold higher than the corresponding values for the widely used substrate, succinyl-Ala-Ala-Pro-Phe-4-nitroanilide, and 200- to 1000-fold higher than the values obtained with succinyl-Ala-Ala-Phe-4-nitroanilide. The favorable effect of the anthraniloyl group as a P4 residue in the substrate sequence Ala-Ala-Phe-4-nitroanilide was assumed to be due to an ability to stiffen S4-P4 interactions. The mechanism proposed is hydrogen bond formation between the phenol group of tyrosine-104 and the amino group of the anthraniloyl moiety. In the spectrophotometric assay with the new substrate, the lower detection limit for subtilisin Carlsberg was 1 nM.