A RAPID METHOD FOR DETERMINATION OF ENDOPROTEINASE SUBSTRATE-SPECIFICITY - SPECIFICITY OF THE 3C PROTEINASE FROM HEPATITIS-A VIRUS

The preferred amino acid residues at the P1' and P2' positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P1' or P2' positions, where a set of 15 or 16 amino acids was introduced. Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termini, which allowed direct sequence analysis by Edman degradation. The relative yield of each amino acid product in the appropriate sequencing cycle gave the amount of each substrate mixture component hydrolyzed. This allowed the simultaneous evaluation of the relative k(cat)/K(m) values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P1' mixture were glycine, alanine, and serine. The enzyme has little specificity at P2'; only arginine and proline peptides were excluded as substrates. This method provides a rapid determination of the preferred residues for a peptide substrate and should be applicable to other endoproteinases.