A rapid method for determination of endoproteinase substrate specificity: Specificity of the 3C proteinase from hepatitis A virus

Journal Article · Sun Dec 15 00:00:00 EST 1991 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.88.24.11510· OSTI ID:5703742

Petithory, J R; Kirsch, J F ^[1]; Masiarz, F R ^[2]; Santi, D V ^[3]; Malcolm, B A ^[4]

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1. Lawrence Berkeley Lab., Berkeley, CA (United States)
2. Chiron Corp., Emeryville, CA (United States) Univ.of California, San Francisco (United States)
3. Univ. of California, San Francisco (United States)
4. Protos Corp., Emeryville, CA (United States)

The preferred amino acid residues at the P{prime}{sub 1} and P{prime}{sub 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{prime}{sub 1} or P{prime}{sub 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 k{sub cat}/K{sub m} values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P{prime}{sub 1} mixture were glycine, alanine, and serine. The enzyme has little specificity at P{prime}{sub 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.

• DOE Contract Number: AC03-76SF00098
• OSTI ID: 5703742
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 88:24; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PEPTIDE HYDROLASES
BIOCHEMICAL REACTION KINETICS
ALANINES
GLYCINE
HEPATITIS
SERINE
SERINE PROTEINASES
SUBSTRATES
VIRUSES
AMINO ACIDS
CARBOXYLIC ACIDS
DIGESTIVE SYSTEM DISEASES
DISEASES
ENZYMES
HYDROLASES
HYDROXY ACIDS
KINETICS
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PROTEINS
REACTION KINETICS
550200* - Biochemistry