Kinetic mechanism of protease inhibition by α1-antitrypsin
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The quaternary state of the human cytomegalovirus (hCMV) protease has been analyzed in relation to its catalysis of peptide hydrolysis. Based on results obtained from steady state kinetics, size exclusion chromatography, and velocity sedimentation, the hCMV protease exists in a monomer-dimer equilibrium. Dimerization of the protease is enhanced by the presence of glycerol and high concentrations of enzyme. Isolation of monomeric and dimeric species eluted from a size exclusion column, followed by immediate assay, identifies the dimer as the active species. Activity measurements conducted with a range of enzyme concentrations are also consistent with a kinetic model in which only the dimeric hCMV protease is active. Using this model, the dissociation constant of the protease is 6.6 μM in 10% glycerol and 0.55 μM in 20% glycerol at 30°C and pH 7.5. The quaternary state of the human cytomegalovirus (hCMV) protease has been analyzed in relation to its catalysis of peptide hydrolysis. Based on results obtained from steady state kinetics, size exclusion chromatography, and velocity sedimentation, the hCMV protease exists in a monomer-dimer equilibrium. Dimerization of the protease is enhanced by the presence of glycerol and high concentrations of enzyme. Isolation of monomeric and dimeric species eluted from a size exclusion column, followed by immediate assay, identifies the dimer as the active species. Activity measurements conducted with a range of enzyme concentrations are also consistent with a kinetic model in which only the dimeric hCMV protease is active. Using this model, the dissociation constant of the protease is 6.6 μM in 10% glycerol and 0.55 μM in 20% glycerol at 30°C and pH 7.5.
Dissociation constant
Sedimentation equilibrium
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The kinetics of the Streptomyces griseus protease‐3‐catalyzed hydrolysis of p ‐nitrophenyl‐acetate have been studied by stopped‐flow techniques with widely varied initial concentrations of enzyme and substrate. The results obtained are consistent with a three‐step mechanism in which there is a rapid equilibration between enzyme and substrate to form a Michaelis complex with a dissociation constant in the order of 10 mM, followed by a moderately rapid formation of an acyl‐enzyme and a rate‐limiting deacylation of this intermediate. Estimated rate constants forthe latter two reaction steps are 15 s −1 and 0.09 s −1 , respectively. The presence of a nucleophile such as methanol results in a partitioning of the deacylation process, reflected by a linear dependence of the maximum steady‐state reaction velocity on the concentration of methanol. This observation provides confirmatory evidence for a rate‐limiting deacylation step in the catalytic mechanism.
Streptomyces griseus
Rate-determining step
Reaction rate
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Dissociation constant
Serine Proteinase Inhibitors
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Linear free energy relationships can be used to link the changes in rate constant for a reaction to changes in the equilibrium caused by alterations in structure. While they have most often been used in the analysis of chemical reactions, they have also been employed to resolve questions in enzymology and protein folding. Here we analyze the reaction of a serpin with a panel of six serine proteinases, and observe that a linear free energy relationship exists between the true second‐order rate constant for reaction, k inh , and the inhibition constant, K I , indicating that formation of the covalent serpin–enzyme complex may be reversible.
Serpin
Folding (DSP implementation)
Serine Proteinase Inhibitors
Free-energy relationship
Constant (computer programming)
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