Mutations that alter the activity of the Rous sarcoma virus protease

B. Grinde, C. E. Cameron, J. Leis, I. T. Weber, A. Wlodawer, H. Burstein, D. Bizub, A. M. Skalka

Research output: Contribution to journalArticle

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Abstract

Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing 'flaps,' and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P, G106V, as well as the triple mutant, H65G, R105P, G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein.

Original languageEnglish (US)
Pages (from-to)9481-9490
Number of pages10
JournalJournal of Biological Chemistry
Volume267
Issue number14
StatePublished - Jan 1 1992

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Viruses
Peptide Hydrolases
HIV-1
Mutation
Glycine
Substrates
Rous sarcoma virus
Histidine
Escherichia coli
Enzymes
Peptides
Structural Models
Flaps
Threonine
Catalysis
Aspartic Acid
Alanine
Rous sarcoma virus protease
Substitution reactions
Crystal structure

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Grinde, B., Cameron, C. E., Leis, J., Weber, I. T., Wlodawer, A., Burstein, H., ... Skalka, A. M. (1992). Mutations that alter the activity of the Rous sarcoma virus protease. Journal of Biological Chemistry, 267(14), 9481-9490.
Grinde, B. ; Cameron, C. E. ; Leis, J. ; Weber, I. T. ; Wlodawer, A. ; Burstein, H. ; Bizub, D. ; Skalka, A. M. / Mutations that alter the activity of the Rous sarcoma virus protease. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 14. pp. 9481-9490.
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Grinde, B, Cameron, CE, Leis, J, Weber, IT, Wlodawer, A, Burstein, H, Bizub, D & Skalka, AM 1992, 'Mutations that alter the activity of the Rous sarcoma virus protease', Journal of Biological Chemistry, vol. 267, no. 14, pp. 9481-9490.

Mutations that alter the activity of the Rous sarcoma virus protease. / Grinde, B.; Cameron, C. E.; Leis, J.; Weber, I. T.; Wlodawer, A.; Burstein, H.; Bizub, D.; Skalka, A. M.

In: Journal of Biological Chemistry, Vol. 267, No. 14, 01.01.1992, p. 9481-9490.

Research output: Contribution to journalArticle

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T1 - Mutations that alter the activity of the Rous sarcoma virus protease

AU - Grinde, B.

AU - Cameron, C. E.

AU - Leis, J.

AU - Weber, I. T.

AU - Wlodawer, A.

AU - Burstein, H.

AU - Bizub, D.

AU - Skalka, A. M.

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Grinde B, Cameron CE, Leis J, Weber IT, Wlodawer A, Burstein H et al. Mutations that alter the activity of the Rous sarcoma virus protease. Journal of Biological Chemistry. 1992 Jan 1;267(14):9481-9490.