A new chromophoric substrate for penicillopepsin and other fungal aspartic proteinases.

The hexapeptide N-alpha-acetylalanylalanyl-lysyl-p- nitrophenylalanylalanylalanylamide has been synthesized and was found to be a good substrate for fungal aspartic proteinases that possess trypsinogen-activating activity, namely penicillopepsin, Rhizopus aspartic proteinase, Endothia aspartic proteinase and the aspartic proteinases from Aspergillus oryzae and Penicillium roqueforti. The peptide is rapidly cleaved between the lysine and p-nitrophenylalanine residues. Calf chymosin and human renin cleave the same bond, but only very slowly. The cleavage is accompanied by an absorbance decrease with a maximum at 296nm (Deltaepsilon -1800m(-1).cm(-1)). Pig pepsin and the aspartic proteinases from two Rhizomucor species cleave the peptide slowly on the carboxy side of p-nitrophenylalanine. For the five enzymes that hydrolysed the peptide rapidly, K(m) values range from 0.16 to 0.42mm and k(cat.) from 6 to 46.6s(-1) at pH 4.5 and 25 degrees C. A comparison of the kinetic parameters of the hexapeptide with those of the dipeptide N-alpha-acetyllysyl-p-nitrophenylalanylamide obtained with penicillopepsin shows that at pH 6.0 the catalytic rate constant k(cat.) is over 5000-fold greater for the hexapeptide, whereas the K(m) values are essentially the same, showing that the catalytic efficiency is strongly dependent on secondary binding. The new substrate with a p-nitrophenylalanine residue in the P'(1) position has advantages over previously used substrates for aspartic proteinases in that it offers a more sensitive spectrophotometric assay that is independent of pH up to 5.5 and can readily be used up to pH 7.0. The presence of lysine makes it very water-soluble. Stopped-flow spectrophotometric experiments with penicillopepsin gave clear evidence that the hydrolysis of the substrate by penicillopepsin is not accompanied by a ;burst' release of p-nitrophenylalanylalanylalanylamide.