Regulation of autoproteolysis of the HIV-1 and HIV-2 proteases with engineered amino acid substitutions

J Biol Chem. 1993 Jun 5;268(16):11939-45.


Autoproteolysis of the retroviral aspartyl proteases is a major obstacle to purification and analysis of these enzymes. A mutagenic approach to rendering autolytic cleavage sites less labile was applied to the primary cleavage site between Leu5 and Trp6 in human immunodeficiency virus-1 (HIV-1) protease. From predictions based on known substrates it was concluded that amino acids Lys or Ser in place of Gln at position 7 would prevent cleavage at the Leu5-Trp6 peptide bond, therefore stabilizing the protein. Autoproteolytic stability was enhanced at least 100-fold by these mutations. At longer time points the protease was degraded at secondary sites which contained adequate substrate sequences but were conformationally restricted. Conversely, a mutation in HIV-2 protease which changed Lys7 to Gln rendered the protein 3-fold less stable and shifted the position of the initial autoproteolytic cleavage from Phe3-Ser4 to Leu5-Trp6. The effects of these mutations demonstrate that small changes in protein sequence can have a major impact on their autoproteolytic stability. The work described here suggests a general method for stabilizing proteases and perhaps other recombinantly produced proteins to autolysis.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Aspartic Acid Endopeptidases / genetics
  • Aspartic Acid Endopeptidases / metabolism*
  • Base Sequence
  • Cloning, Molecular
  • Endopeptidases / genetics
  • Endopeptidases / metabolism
  • Escherichia coli / genetics
  • HIV Protease / genetics
  • HIV Protease / metabolism*
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • HIV-2 / enzymology*
  • HIV-2 / genetics
  • Kinetics
  • Leucine
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • Protein Engineering
  • Recombinant Proteins / metabolism
  • Simian Immunodeficiency Virus / enzymology
  • Simian Immunodeficiency Virus / genetics
  • Substrate Specificity
  • Tryptophan


  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • Tryptophan
  • Endopeptidases
  • Aspartic Acid Endopeptidases
  • HIV Protease
  • p16 protease, Human immunodeficiency virus 2
  • Leucine