HUMAN-IMMUNODEFICIENCY-VIRUS, TYPE-1 PROTEASE SUBSTRATE-SPECIFICITY IS LIMITED BY INTERACTIONS BETWEEN SUBSTRATE AMINO-ACIDS BOUND IN ADJACENT ENZYME SUBSITES

The specificity of the retroviral protease is determined by the abilit y of substrate amino acid side chains to bind into eight individual su bsites within the enzyme, Although the subsites are able to act somewh at independently in selection of amino acid side chains that fit into each pocket, significant interactions exist between individual subsite s that substantially limit the number of cleavable amino acid sequence s, The substrate peptide binds within the enzyme in an extended anti-p arallel beta sheet conformation with substrate amino acid side chains adjacent in the linear sequence extending in opposite directions in th e enzyme-substrate complex, From this geometry, we have defined both c is and trans steric interactions, which have been characterized by a s teady state kinetic analysis of human immunodeficiency virus, type-1 p rotease using a series of peptide substrates that are derivatives of t he avian leukosis/sarcoma virus nucleocapsid-protease cleavage site, T hese peptides contain both single and double amino acid substitutions in seven positions of the minimum length substrate required by the ret roviral protease for specific and efficient cleavage, Steady state kin etic data from the single amino acid substituted peptides were used to predict effects on protease-catalyzed cleavage of corresponding doubl e substituted peptide substrates, The calculated Gibbs' free energy ch anges were compared with actual experimental values in order to determ ine how the fit of a substrate amino acid in one subsite influences th e fit of amino acids in adjacent subsites, Analysis of these data show s that substrate specificity is limited by steric interactions between pairs of enzyme subsites, Moreover, certain enzyme subsites are relat ively tolerant of substitutions in the substrate and exert little effe ct on adjacent subsites, whereas others are more restrictive and have marked influence on adjacent cis and trans subsites.