HIV-1 protease dimer stability and enzyme kinetics studied by high-pressure methods

Kód výsledku v IS VaVaI

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Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

HIV-1 protease dimer stability and enzyme kinetics studied by high-pressure methods

Popis výsledku v původním jazyce

High-pressure methods have become a useful tool of biomolecular research during the last decades. Application of high pressure to biomolecules allows us to study various structural changes that are hardly observable at the atmospheric pressure. It is, therefore, possible to observe processes like protein unfolding, dissociation of oligomeric proteins or protein and its non-protein ligand, change of the rate of enzymatic reactions, etc., and to deduce the values of their thermodynamics and kinetics parameters at atmospheric pressure. HIV-1 protease is a proteolytic enzyme of HIV virus active as a homodimer of two subunits of 99 amino acids each. This enzyme was intensively studied in the past in many points of view, including its structure, enzyme kinetics and dimer dissociation, often within various pharmaceutical and drug-design research projects. Due to the high dimer stability, determination of the dissociation constant Kd is rather a tricky problem that provided different, often rather inconsistent, results in the past. As high pressure supports the dimer dissociation, it is quite a helpful instrument to study this phenomenon. We have determined the dissociation constant Kd of HIV-1 protease by the method based on the measurement of the intrinsic tryptophan fluorescence under varying pressure. The atmospheric-pressure value of Kd was determined from the dependence of the inflection point of the measured curves on the pressure, together with the volume change of dimer dissociation DVd. The determined value of Kd = 0.92 µM compares reasonably with other results obtained by conventional methods and DVd = –32.5 ml mol-1 is consistent with similar systems studied under high pressure. Molecular-dynamics simulation of the enzyme at varying pressures revealed that the volume change originates from lower density of water molecules inside the hydrophobic active-site cavity of dimer and causes an approximately linear decrease of the pKd value throughout a substantial part of the pressure interval. High-pressure fluorescence assays also revealed that the dissociated molecules of HIV-1 protease unfold above the pressure of 250 MPa, while an inhibitor-stabilized dimer does not, which confirms the hypothesis of higher stability of the dimer in comparison with monomer. In order to describe the influence of the dimer-monomer equilibrium on both the stability and catalytic activity of the enzyme, a comparison was carried out between the wt-HIV-1 protease and its analog consisting of the covalently linked monomers that is unable to dissociate. At this enzyme, the unfolding occurs only at pressures above 400 MPa. The rate of the catalyzed substrate cleavage is comparable at the atmospheric pressure for both the enzymes, but decreases more rapidly for the wt enzyme than for the covalently linked dimer. These observations are coherent with the stabilizing effect of dimer formation. In addition, the pressure dependence enables the separation of the influence of dimer dissociation from the pressure effects on the kinetics constants, Km and kcat, and allows us to determine their pressure dependencies and the atmospheric-ressure value of Kd in a more sensitive way.

Název v anglickém jazyce

HIV-1 protease dimer stability and enzyme kinetics studied by high-pressure methods

Popis výsledku anglicky

High-pressure methods have become a useful tool of biomolecular research during the last decades. Application of high pressure to biomolecules allows us to study various structural changes that are hardly observable at the atmospheric pressure. It is, therefore, possible to observe processes like protein unfolding, dissociation of oligomeric proteins or protein and its non-protein ligand, change of the rate of enzymatic reactions, etc., and to deduce the values of their thermodynamics and kinetics parameters at atmospheric pressure. HIV-1 protease is a proteolytic enzyme of HIV virus active as a homodimer of two subunits of 99 amino acids each. This enzyme was intensively studied in the past in many points of view, including its structure, enzyme kinetics and dimer dissociation, often within various pharmaceutical and drug-design research projects. Due to the high dimer stability, determination of the dissociation constant Kd is rather a tricky problem that provided different, often rather inconsistent, results in the past. As high pressure supports the dimer dissociation, it is quite a helpful instrument to study this phenomenon. We have determined the dissociation constant Kd of HIV-1 protease by the method based on the measurement of the intrinsic tryptophan fluorescence under varying pressure. The atmospheric-pressure value of Kd was determined from the dependence of the inflection point of the measured curves on the pressure, together with the volume change of dimer dissociation DVd. The determined value of Kd = 0.92 µM compares reasonably with other results obtained by conventional methods and DVd = –32.5 ml mol-1 is consistent with similar systems studied under high pressure. Molecular-dynamics simulation of the enzyme at varying pressures revealed that the volume change originates from lower density of water molecules inside the hydrophobic active-site cavity of dimer and causes an approximately linear decrease of the pKd value throughout a substantial part of the pressure interval. High-pressure fluorescence assays also revealed that the dissociated molecules of HIV-1 protease unfold above the pressure of 250 MPa, while an inhibitor-stabilized dimer does not, which confirms the hypothesis of higher stability of the dimer in comparison with monomer. In order to describe the influence of the dimer-monomer equilibrium on both the stability and catalytic activity of the enzyme, a comparison was carried out between the wt-HIV-1 protease and its analog consisting of the covalently linked monomers that is unable to dissociate. At this enzyme, the unfolding occurs only at pressures above 400 MPa. The rate of the catalyzed substrate cleavage is comparable at the atmospheric pressure for both the enzymes, but decreases more rapidly for the wt enzyme than for the covalently linked dimer. These observations are coherent with the stabilizing effect of dimer formation. In addition, the pressure dependence enables the separation of the influence of dimer dissociation from the pressure effects on the kinetics constants, Km and kcat, and allows us to determine their pressure dependencies and the atmospheric-ressure value of Kd in a more sensitive way.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

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Návaznosti

V - Vyzkumna aktivita podporovana z jinych verejnych zdroju

Rok uplatnění

2017

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů