Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease.

DiIanni, Carolyn L.; Davis, Lenora J.; Holloway, M. K.; Herber, Wayne K.; Darke, Paul L.; Kohl, Nancy E.; Dixon, Richard A. F.

doi:10.1016/s0021-9258(17)44909-x

Cited by 66 publications

(14 citation statements)

References 40 publications

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“…As controls, incubation of the active protease with other proteins not expected to participate in specific subunit exchange, such as lysozyme, had no effect on activity. Also, the single-chain D126N form of the protease (Dilanni et al, 1990a), which cannot dissociate to monomers, also had no effect on the activity (data not shown).…”

Section: Resultsmentioning

confidence: 95%

“…These results suggested that the dimeric enzyme was dissociating into monomers in dilute solutions during preincubation. Indeed, when a form of the enzyme which cannot dissociate into subunits (Dilanni et al, 1990a) was substituted for the wild-type enzyme in the experiments described above, the fluorescence changes were not affected by preincubation of the enzyme. Preincubation of this single polypeptide form of the HIV-1 protease produced fluorescence changes the same as those shown in Figure 1A for the no-preincubation case (data not shown).…”

Section: Resultsmentioning

confidence: 97%

“…We have previously shown that changing one of the two active-site Asp-25 residues to an Asn is sufficient for total inactivation of the HIV-1 protease, through use of a single polypeptide form of the enzyme, D126N, in which the two subunits are covalently linked (Dilanni et al, 1990a). The loss of activity upon mixing of the active enzyme and D25N mutant is then reasonably interpreted as the formation of an inactive heterodimer according to Scheme 2.…”

Section: Discussionmentioning

confidence: 99%

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Dissociation and association of the HIV-1 protease dimer subunits: Equilibria and rates

Darke¹,

Jordan²,

Hall³

et al. 1994

Biochemistry

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The kinetics and equilibrium properties were investigated for the interconversion between the active dimer of human immunodeficiency virus 1 (HIV-1) protease and its inactive monomeric subunits. The equilibrium dissociation constant (Kd) of the dimeric protease as well as the monomer association rate were obtained by monitoring the fluorescence change of an active-site-directed fluorescent probe (L-737244) upon its binding to the protease. The Kd of the HIV-1 protease is strongly pH dependent. At pH 5.5 where the enzyme is most active catalytically, the extrapolated values of Kd are 0.75 and 3.4 nM at 30 and 37 degrees C, respectively. The rate constant for HIV-1 monomer association, approximately 4 x 10(5) M-1 s-1, is within the range commonly observed for protein-protein interactions. Dimer dissociation was further scrutinized in the presence of an inactive, point mutant form of the enzyme. As a result of subunit exchange between the native and mutant enzymes and the formation of an inactive heterodimer, there was a time-dependent decrease in the activity of the native protease. Enzyme activity could be reinstated with the addition of an active-site-directed inhibitor (L-365862) which selectively binds active dimers. The rate of dimer dissociation was found to also decrease with pH. At pH 5.5 and 30 degrees C, the half-life for subunit dissociation is about 0.5 h. The slow dissociation, coupled with the high stability for dimer association, attests to the importance of allowing sufficient time for dimer-monomer equilibration in kinetic assays in order to avoid reaching erroneous conclusions in studies of dimer dissociation.

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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

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Dissociation and association of the HIV-1 protease dimer subunits: Equilibria and rates

Darke¹,

Jordan²,

Hall³

et al. 1994

Biochemistry

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“…We have shown that both HIV-1 PR and SIV PR are inactivated and unfolded at urea concentrations of 2-4 M, with transition midpoints ranging from 1.9 to 3.1 M urea. Dilanni et al (1990a) have reported unfolding of wild-type and mutant forms of HI V-1 PR at similar urea concentrations by using nondenaturing electrophoresis. In contrast, the monomeric aspartic protease, porcine pepsin, has been reported to be active at 6 M urea (Steinhardt, 1938;Fruton, 1976).…”

Section: Discussionmentioning

confidence: 99%

Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases

et al. 1992

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The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0

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“…While thepol gene of HIV codes for the protease as a single chain polypeptide which contains 99 amino acids, the protease functional unit is a homodimer (Meek et al, 1989;Cheng et al, 1990;Dilanni et al, 1990;Babe et al, 1991;Grant et al, 1992) . Crystal structure determination has shown the dimeric HIV protease to be structurally homologous to members of the aspartic protease family which includes, among others, pepsin, renin, and cathepsin D. The active site of these proteases contains two aspartic acid residues and a highly t This work is supported by NIH Grant Al 26762.…”

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confidence: 99%

Proteolytic Processing Mechanisms of a Miniprecursor of the Aspartic Protease of Human Immunodeficiency Virus Type 1

Co¹,

Koelsch²,

Lin³

et al. 1994

Biochemistry

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The infectivity of the human immunodeficiency virus (HIV) depends upon correct proteolytic processing of viral polyprotein precursors, the Pr55gag and Pr160gag-pol polyproteins. The processing is mediated spontaneously by the viral protease unit (PR) contained within the Pr160gag-pol precursor. However, little is known about the mechanism of this process. The expression in Escherichia coli and the isolation of a 14-kDa HIV-1 PR "miniprecursor" with Ala28 mutated to serine has permitted study of the mechanism for cleavage at the N-terminus of the protease. The miniprecursor is active against a synthetic peptide substrate, and its specific activity is near that of the mutant mature protease. The rate of conversion of radiolabeled precursor to mature protease is quantitated by measuring the amounts of the two radiolabeled proteins separated by SDS-PAGE. The apparent first-order conversion rate constant, kapp, is dependent on miniprecursor concentration indicating a second-order reaction and suggesting an interdimeric processing mechanism. A significant first-order rate constant is observed when the plot of kapp versus initial precursor concentration is extrapolated to zero. This observation suggests the presence of an alternative processing mechanism involving a single active precursor dimer. The presence of both mechanisms is an advantage for the virus to ensure processing under various conditions.

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Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease.

Cited by 66 publications

References 40 publications

Dissociation and association of the HIV-1 protease dimer subunits: Equilibria and rates

Dissociation and association of the HIV-1 protease dimer subunits: Equilibria and rates

Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases

Proteolytic Processing Mechanisms of a Miniprecursor of the Aspartic Protease of Human Immunodeficiency Virus Type 1

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