REGULATION OF AUTOPROTEOLYSIS OF THE HIV-1 AND HIV-2 PROTEASES WITH ENGINEERED AMINO-ACID SUBSTITUTIONS

Autoproteolysis of the retroviral aspartyl proteases is a major obstac le 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 positi on 7 would prevent cleavage at the Leu5-Trp6 peptide bond, therefore s tabilizing the protein. Autoproteolytic stability was enhanced at leas t 100-fold by these mutations. At longer time points the protease was degraded at secondary sites which contained adequate substrate sequenc es 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 cleava ge from Phe3-Ser4 to Leu5-Trp6. The effects of these mutations demonst rate that small changes in protein sequence can have a major impact on their autoproteolytic stability. The work described here suggests a g eneral method for stabilizing proteases and perhaps other recombinantl y produced proteins to autolysis.