Dissociative inhibition of dimeric enzymes: Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide

Zhong-Yin Zhang, Roger A. Poorman, Linda L. Maggiora, Robert L. Heinrikson, Ferenc J. Kézdy

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Abstract

Human immunodeficiency virus 1 (HIV-1) protease is an aspartyl protease composed of two identical protomers linked by a four-stranded antiparallel β-sheet consisting of the NH2- and COOH-terminal segments (Weber, I. T. (1990) J. Biol. Chem. 265, 10492-10496). Kinetic analysis of the HIV-1 protease-catalyzed hydrolysis of a fluorogenic substrate demonstrates that the enzyme is an obligatory dimer. At pH = 5.0, 0.1 M sodium acetate, 1 M NaCl, 1 mM EDTA buffer, 37 °C, the equilibrium dissociation constant, Kd = 3.6 ± 1.9 nM. We found that the tetrapeptide Ac-Thr-Leu-Asn-Phe-COOH, corresponding to the COOH-terminal segment of the enzyme, is an excellent inhibitor of the enzyme. Kinetic analysis shows that the inhibitor binds to the inactive protomers and prevents their association into the active dimer (dissociative inhibition). The dissociative nature of this inhibition is consistent with the results obtained from sedimentation equilibrium experiments in which the apparent molecular weight of the enzyme was observed to be 20,800 ± 1,500 and 12,100 ± 300, in the absence and presence of the COOH-terminal tetrapeptide, respectively. The dissociation constant of the protomer-inhibitor complex is Ki = 45.1 ± 1.8 μM. This is the first kinetic analysis and direct experimental demonstration of noncovalent dissociative inhibition.

Original languageEnglish (US)
Pages (from-to)15591-15594
Number of pages4
JournalJournal of Biological Chemistry
Volume266
Issue number24
StatePublished - 1991
Externally publishedYes

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Enzyme inhibition
Enzyme kinetics
Protein Subunits
Viruses
HIV-1
Peptide Hydrolases
Dimers
Enzymes
Aspartic Acid Proteases
Sodium Acetate
Enzyme Inhibitors
Fluorescent Dyes
Sedimentation
Edetic Acid
Hydrolysis
Buffers
Demonstrations
Molecular Weight
Molecular weight
Association reactions

ASJC Scopus subject areas

  • Biochemistry

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Dissociative inhibition of dimeric enzymes : Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide. / Zhang, Zhong-Yin; Poorman, Roger A.; Maggiora, Linda L.; Heinrikson, Robert L.; Kézdy, Ferenc J.

In: Journal of Biological Chemistry, Vol. 266, No. 24, 1991, p. 15591-15594.

Research output: Contribution to journalArticle

Zhang, Zhong-Yin ; Poorman, Roger A. ; Maggiora, Linda L. ; Heinrikson, Robert L. ; Kézdy, Ferenc J. / Dissociative inhibition of dimeric enzymes : Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 24. pp. 15591-15594.
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