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 K m = 0.004 mM, k cat = 104 s −1, and those for subtilisin BPN′ are K m = 0.020 mM, k cat = 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 P 4 residue in the substrate sequence Ala Ala Phe-4-nitroanilide was assumed to be due to an ability to stiffen S 4-P 4 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 n m.