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 P'1 and P'2 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 P'1 or P'2 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 kcat/Km values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P'1 mixture were glycine, alanine, and serine. The enzyme has little specificity at P'2; 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.
Petithory
J R
JR
Department of Molecular and Cell Biology, University of California, Berkeley.
Masiarz
F R
FR
Kirsch
J F
JF
Santi
D V
DV
Malcolm
B A
BA
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006507:Hepatovirus / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins
D000596:Amino Acids; D009842:Oligopeptides; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases