Dataset Information

Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor.

ABSTRACT: The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MPro^WT) was assessed using a predominantly monomeric mutant (MPro^M). Rates of MPro^WT and MPro^M catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MPro^WT, to MPro^M leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.

SUBMITTER: Nashed NT

PROVIDER: S-EPMC8888643 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor.

Nashed Nashaat T NT Aniana Annie A Ghirlando Rodolfo R Chiliveri Sai Chaitanya SC Louis John M JM

Communications biology 20220301 1

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Rates of MProWT and MProM catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MProWT, to MProM leads to an increase in the dimer population and catalytic ac ...[more]

PMID: 35233052

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Project description:Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

Dataset Information

Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor.

Publications

Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor.

Similar Datasets

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