Document 0153
 DOCN  M94A0153
 TI    The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis
       sites and generation of a stable mutant with retained kinetic
       properties.
 DT    9412
 AU    Mildner AM; Rothrock DJ; Leone JW; Bannow CA; Lull JM; Reardon IM;
       Sarcich JL; Howe WJ; Tomich CS; Smith CW; et al; Biochemistry Unit,
       Upjohn Laboratories, Kalamazoo, Michigan; 49001.
 SO    Biochemistry. 1994 Aug 16;33(32):9405-13. Unique Identifier : AIDSLINE
       MED/94347706
 AB    Site-directed mutagenesis of autolysis sites in the human
       immunodeficiency virus type 1 (HIV-1) protease was applied in an
       analysis of enzyme specificity; the protease served, therefore, as both
       enzyme and substrate in this study. Inspection of natural substrates of
       all retroviral proteases revealed the absence of beta-branched amino
       acids at the P1 site and of Lys anywhere from P2 through P2'.
       Accordingly, several mutants of the HIV-1 protease were engineered in
       which these excluded amino acids were substituted at their respective P
       positions at the three major sites of autolysis in the wild-type
       protease (Leu5-Trp6, Leu33-Glu34, and Leu63-Ile64), and the mutant
       enzymes were evaluated in terms of their resistance to autodegradation.
       All of the mutant HIV-1 proteases, expressed as inclusion bodies in
       Escherichia coli, were enzymatically active after refolding, and all
       showed greatly diminished rates of cleavage at the altered autolysis
       sites. Some, however, were not viable enzymatically because of poor
       physical characteristics. This was the case for mutants having Lys
       replacements of Glu residues at P2' and for another in which all three
       P1 leucines were replaced by Ile. However, one of the mutant proteases,
       Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the
       physical properties, specificity, and susceptibility to inhibition of
       the wild-type enzyme. Q7K/L33I/L63I should find useful application as a
       stable surrogate of the HIV-1 protease. Overall, our results can be
       interpreted relative to a model in which the active HIV-1 protease dimer
       is in equilibrium with monomeric, disordered species which serve as the
       substrates for autolysis.
 DE    Amino Acid Sequence  Comparative Study  HIV
       Protease/GENETICS/*METABOLISM  HIV Protease Inhibitors/PHARMACOLOGY
       HIV-1/*ENZYMOLOGY  Models, Molecular  Molecular Sequence Data
       Mutagenesis, Site-Directed  Oligopeptides/METABOLISM  Protein
       Conformation  *Protein Processing, Post-Translational
       Structure-Activity Relationship  Substrate Specificity  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).