The effect of salt on the Michaelis Menten constant of the HIV‐1 protease correlates with the Hofmeister series

Wondrak, Ewald M.; Louis, John M.; Oroszlan, Stephen

doi:10.1016/0014-5793(91)80327-y

Cited by 68 publications

(53 citation statements)

References 17 publications

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“…One factor of the promotion of catalysis may be the salting in of substrate to the enzyme surface, which is supported by the lower K m at higher ionic strength (3,4). We propose that an additional factor to be considered is the conformational stability of the enzyme.…”

Section: Discussionmentioning

confidence: 97%

“…The activation has been explained by assuming that the substrates are "salting in" to the active site of the enzyme (3,4). Ionic strength may also modify the conformational stability of the protein.…”

Section: Discussionmentioning

confidence: 99%

“…1 and 2). High salt concentration was found to enhance the catalytic activity, primarily by lowering the Michaelis constant (K m ), and it was suggested that the salting-out effect of NaCl decreased K m and increased k cat /K m , the specificity rate constant (3,4). We have pointed out that K m values may be markedly dependent on pH, while the rate enhancement by salt is practically independent of K m and pH (5).…”

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confidence: 99%

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Conformational Stability and Catalytic Activity of HIV-1 Protease Are Both Enhanced at High Salt Concentration

Szeltner

Polgár

1996

Journal of Biological Chemistry

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The activity of human immunodeficiency virus protease is markedly increased at elevated salt concentration. The structural basis of this effect has been explored by several independent methods by using both the wild-type enzyme and its triple mutant (Q7K/L33I/ L63I) (Mildner, and the transition midpoint (D1 ⁄2 ) between the folded and unfolded states increase, indicating that the salt stabilizes the enzyme structure. These equilibrium data are supported by kinetic studies on the urea-mediated unfolding by measuring fluorescence change, red shifting in the maximum of the emission spectrum, and farand near-UV CD. The salt effects observed in urea-mediated unfolding reactions prevail upon heat denaturation. All these findings support the existence of a twostate equilibrium between the folded and unfolded proteins. The pH dependence of fluorescence intensity indicated that the conformation of human immunodeficiency virus type 1 protease should change in the catalytically competent pH region. It is concluded that preferential hydration stabilizes the protease structure in the presence of salt, providing entropic contribution to enhance the catalytic activity.AThe protease encoded by HIV-1 1 is involved in the specific processing of large viral polyproteins into individual structural proteins and enzymes. This prompted extensive investigations on HIV-1 protease as a potential therapeutic target of AIDS. The enzyme is a member of the aspartic protease family and exists as homodimer of identical polypeptide chains, each consisting of 99 amino acid residues (cf. Refs. 1 and 2). High salt concentration was found to enhance the catalytic activity, primarily by lowering the Michaelis constant (K m ), and it was suggested that the salting-out effect of NaCl decreased K m and increased k cat /K m , the specificity rate constant (3, 4). We have pointed out that K m values may be markedly dependent on pH, while the rate enhancement by salt is practically independent of K m and pH (5). Therefore, other factors, for example the conformational stability of the protein, may also be important. Indeed, raising the NaCl concentration frequently stabilizes the protein structure by preferential hydration (6). However, the effects of ionic strength on the conformational stability of HIV-1 protease have not yet been studied. To reveal whether or not the enhanced catalytic activity is associated with a more stable structure, we have examined the stability of the enzyme against urea, pH, and heat denaturation at different salt concentrations. A mutant enzyme designed to resist autolysis (7) has been used in most experiments. EXPERIMENTAL PROCEDURESHIV-1 Protease-The enzyme was expressed in Escherichia coli and purified as described (5). The construction containing three mutations (Q7K/L33I/L63I) in the HIV-1 protease was kindly supplied by Dr. Tomasselli (7), and the enzyme was purified from inclusion bodies with the same method as used for the wild-type protease. The protein concentration was determined at 280 nm (5). The activity of enzym...

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Conformational Stability and Catalytic Activity of HIV-1 Protease Are Both Enhanced at High Salt Concentration

Szeltner

Polgár

1996

Journal of Biological Chemistry

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“…[10][11][12][13][14][15] This is in contrast to the case of human immunodeficiency virus type-1 (HIV-1) protease, in which activation is same as in the Hofmeister series. 26,27) Based on this evidence, we have pointed out that salt-induced activation of thermolysin cannot be explained only by a reduction in dielectric constant of the reaction medium, and that it is in part the result of direct interactions of enzymes with ions. [10][11][12][13][14][15] Mutation Asp150!Glu increases activity.…”

Section: Discussionmentioning

confidence: 99%

Effects of Mutations of Thermolysin, Asn116 to Asp and Asp150 to Glu, on Salt-Induced Activation and Stabilization

Menach

Yasukawa

Inouye

2013

Bioscience, Biotechnology, and Biochemistry

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“…Altered composition of ions has been reported to directly affect activation of proteins involved in apoptosis (cytochrome c, proteases, nucleases etc), as well as formation of the apoptosome, affecting the ratio between pro-and antiapoptotic proteins Hampton et al, 1998;Hughes et al, 1997;Karki et al, 2007;Koeberle et al, 2010;Strickland et al, 1991;Thompson et al, 2001;Wondrak et al, 1991).…”

Section: Plasma Membrane-bound Voltage-gated Ion Channels Are Importamentioning

confidence: 99%

The role of ion channels and intracellular metal ions in apoptosis of Xenopus oocytes

Englund¹

2014

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Apoptosis is one type of programmed cell death, important during tissue development and to maintain the tissue homeostasis. Apoptosis comprises a complex network of internal signaling pathways, and an important part of this signaling network is the action of voltage‐gated ion channels. The aim of this thesis was to explore the role of ion channels and the role of intracellular metal ions during apoptosis in Xenopus laevis oocytes. The reasons for using these oocytes are that they are large, robust, easy to handle, and easy to study electrophysiologically. Apoptosis was induced either chemically by incubation of the oocytes in staurosporine (STS) or mechanically by centrifugation of the oocytes. Ion currents were measured by a two‐electrode voltage clamp technique, intracellular ion concentrations were measured either directly by in‐house developed K+‐selective microelectrodes or indirectly by the electrophysiological technique, and apoptosis was measured by caspase‐3 activation. Paper I describes that the intracellular K+ concentration was reduced by about 30 % during STS‐induced apoptosis. However, this reduction was prevented by excessive expression of exogenous ion channels. Despite the magnitude of the intracellular K+ concentration, either normal or reduced level, the oocytes displayed normal signs of apoptosis, suggesting that the intracellular K+ reduction was not required for the apoptotic process. Because the intracellular K+ concentration was not critical for apoptosis we searched for other ion fluxes by exploring the electrophysiological properties of X. laevis oocytes. Paper II, describes a non‐inactivating Na+ current activated at positive membrane voltages that was upregulated by a factor of five during STS‐induced apoptosis. By preventing influx of Na+, the apoptotic signaling network involving capsase‐3 was prevented. To molecularly identify this voltage‐gated Na channel, the X. tropicalis genome and conserved regions of the human SCNA genes were used as a map. Paper III, shows that the voltage‐gated Na channel corresponds to the SCN2A gene ortholog and that supression of this SCN2A ortholog using miRNA prevented cell death. In conclusion, this thesis work demonstrated that a voltage‐gated Na channel is critical for the apoptotic process in X. laevis oocytes by increasing the intracellular Na+ concentration

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The effect of salt on the Michaelis Menten constant of the HIV‐1 protease correlates with the Hofmeister series

Cited by 68 publications

References 17 publications

Conformational Stability and Catalytic Activity of HIV-1 Protease Are Both Enhanced at High Salt Concentration

Conformational Stability and Catalytic Activity of HIV-1 Protease Are Both Enhanced at High Salt Concentration

Effects of Mutations of Thermolysin, Asn116 to Asp and Asp150 to Glu, on Salt-Induced Activation and Stabilization

The role of ion channels and intracellular metal ions in apoptosis of Xenopus oocytes

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