Human immunodeficiency virus, type 1 protease substrate specificity is limited by interactions between substrate amino acids bound in adjacent enzyme subsites

Todd W. Ridky, Craig Eugene Cameron, John Cameron, Jonathan Leis, Terry Copeland, Alexander Wlodawer, Irene T. Weber, Robert W. Harrison

Research output: Contribution to journalArticle

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Abstract

The specificity of the retroviral protease is determined by the ability of substrate amino acid side chains to bind into eight individual subsites within the enzyme. Although the subsites are able to act somewhat independently in selection of amino acid side chains that fit into each pocket, significant interactions exist between individual subsites that substantially limit the number of clearable amino acid sequences. The substrate peptide binds within the enzyme in an extended anti-parallel β sheet conformation with substrate amino acid side chains adjacent in the linear sequence extending in opposite directions in the enzyme-substrate complex. From this geometry, we have defined both cis and trans steric interactions, which have been characterized by a steady state kinetic analysis of human immunodeficiency virus, type-1 protease using a series of peptide substrates that are derivatives of the avian leukosis/sarcoma virus nucleocapsid-protease cleavage site. These peptides contain both single and double amino acid substitutions in seven positions of the minimum length substrate required by the retroviral protease for specific and efficient cleavage. Steady state kinetic data from the single amino acid substituted peptides were used to predict effects on protease-catalyzed cleavage of corresponding double substituted peptide substrates. The calculated Gibbs' free energy changes were compared with actual experimental values in order to determine how the fit of a substrate amino acid in one subsite influences the fit of amino acids in adjacent subsites. Analysis of these data shows that substrate specificity is limited by steric interactions between pairs of enzyme subsites. Moreover, certain enzyme subsites are relatively tolerant of substitutions in the substrate and exert little effect on adjacent subsites, whereas others are more restrictive and have marked influence on adjacent cis and trans subsites.

Original languageEnglish (US)
Pages (from-to)4709-4717
Number of pages9
JournalJournal of Biological Chemistry
Volume271
Issue number9
DOIs
StatePublished - Mar 1 1996

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Substrate Specificity
Viruses
HIV-1
Peptide Hydrolases
Amino Acids
Substrates
Enzymes
Peptides
Alpharetrovirus
Nucleocapsid
Amino Acid Substitution
Substitution reactions
Amino Acid Sequence
Kinetics
Gibbs free energy
Conformations
Derivatives
Geometry

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Ridky, Todd W. ; Cameron, Craig Eugene ; Cameron, John ; Leis, Jonathan ; Copeland, Terry ; Wlodawer, Alexander ; Weber, Irene T. ; Harrison, Robert W. / Human immunodeficiency virus, type 1 protease substrate specificity is limited by interactions between substrate amino acids bound in adjacent enzyme subsites. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 9. pp. 4709-4717.
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abstract = "The specificity of the retroviral protease is determined by the ability of substrate amino acid side chains to bind into eight individual subsites within the enzyme. Although the subsites are able to act somewhat independently in selection of amino acid side chains that fit into each pocket, significant interactions exist between individual subsites that substantially limit the number of clearable amino acid sequences. The substrate peptide binds within the enzyme in an extended anti-parallel β sheet conformation with substrate amino acid side chains adjacent in the linear sequence extending in opposite directions in the enzyme-substrate complex. From this geometry, we have defined both cis and trans steric interactions, which have been characterized by a steady state kinetic analysis of human immunodeficiency virus, type-1 protease using a series of peptide substrates that are derivatives of the avian leukosis/sarcoma virus nucleocapsid-protease cleavage site. These peptides contain both single and double amino acid substitutions in seven positions of the minimum length substrate required by the retroviral protease for specific and efficient cleavage. Steady state kinetic data from the single amino acid substituted peptides were used to predict effects on protease-catalyzed cleavage of corresponding double substituted peptide substrates. The calculated Gibbs' free energy changes were compared with actual experimental values in order to determine how the fit of a substrate amino acid in one subsite influences the fit of amino acids in adjacent subsites. Analysis of these data shows that substrate specificity is limited by steric interactions between pairs of enzyme subsites. Moreover, certain enzyme subsites are relatively tolerant of substitutions in the substrate and exert little effect on adjacent subsites, whereas others are more restrictive and have marked influence on adjacent cis and trans subsites.",
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Human immunodeficiency virus, type 1 protease substrate specificity is limited by interactions between substrate amino acids bound in adjacent enzyme subsites. / Ridky, Todd W.; Cameron, Craig Eugene; Cameron, John; Leis, Jonathan; Copeland, Terry; Wlodawer, Alexander; Weber, Irene T.; Harrison, Robert W.

In: Journal of Biological Chemistry, Vol. 271, No. 9, 01.03.1996, p. 4709-4717.

Research output: Contribution to journalArticle

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AU - Ridky, Todd W.

AU - Cameron, Craig Eugene

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