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Product inhibition in native-state proteolysis.

Kasper JR, Andrews EC, Park C - PLoS ONE (2014)

Bottom Line: First-order kinetics has been assumed for the analysis of native-state proteolysis.By analyzing the kinetics of proteolysis with varying concentrations of substrate proteins and also with cleavage products, we found that the deviation from first-order kinetics results from product inhibition.A kinetic model including competitive product inhibition agrees well with the proteolysis time course and allows us to determine the uninhibited rate constant for proteolysis as well as the apparent inhibition constant.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, United States of America.

ABSTRACT
The proteolysis kinetics of intact proteins by nonspecific proteases provides valuable information on transient partial unfolding of proteins under native conditions. Native-state proteolysis is an approach to utilize the proteolysis kinetics to assess the energetics of partial unfolding in a quantitative manner. In native-state proteolysis, folded proteins are incubated with nonspecific proteases, and the rate of proteolysis is determined from the disappearance of the intact protein. We report here that proteolysis of intact proteins by nonspecific proteases, thermolysin and subtilisin deviates from first-order kinetics. First-order kinetics has been assumed for the analysis of native-state proteolysis. By analyzing the kinetics of proteolysis with varying concentrations of substrate proteins and also with cleavage products, we found that the deviation from first-order kinetics results from product inhibition. A kinetic model including competitive product inhibition agrees well with the proteolysis time course and allows us to determine the uninhibited rate constant for proteolysis as well as the apparent inhibition constant. Our finding suggests that the likelihood of product inhibition must be considered for quantitative assessment of proteolysis kinetics.

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Dependence of the apparent proteolysis rate constants (kp) on concentration of substrate proteins.(A) Proteolysis of DHFR by 80 µg/ml thermolysin. (B) Proteolysis of RNase H by 200 µg/ml thermolysin. (C) Proteolysis of DHFR by 40 µg/ml subtilisin. Error bars show the standard errors from curve-fitting to a first-order rate equation.
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pone-0111416-g001: Dependence of the apparent proteolysis rate constants (kp) on concentration of substrate proteins.(A) Proteolysis of DHFR by 80 µg/ml thermolysin. (B) Proteolysis of RNase H by 200 µg/ml thermolysin. (C) Proteolysis of DHFR by 40 µg/ml subtilisin. Error bars show the standard errors from curve-fitting to a first-order rate equation.

Mentions: To verify that our proteolysis condition satisfies pseudo-first-order kinetics, we determined the apparent first-order rate constants for proteolysis of DHFR by thermolysin at varying concentrations of DHFR (0.050–0.50 mg/mL) (Figure 1A). Interestingly, we observed that proteolysis of DHFR by thermolysin is slower with higher concentrations of DHFR. A ten-fold increase in DHFR concentration from 50 to 500 µg/ml resulted in a seven-fold decrease in the apparent rate constant. This dependence of the apparent kinetic constant on the substrate concentration suggests that the reaction does not satisfy the assumption of pseudo-first-order kinetics in which the apparent rate constant should depend only on the enzyme concentration. Fitting of the plot of remaining intact protein versus time to a first-order rate equation also shows non-random residuals, which indicates slight deviation from first-order kinetics (Figure S1).


Product inhibition in native-state proteolysis.

Kasper JR, Andrews EC, Park C - PLoS ONE (2014)

Dependence of the apparent proteolysis rate constants (kp) on concentration of substrate proteins.(A) Proteolysis of DHFR by 80 µg/ml thermolysin. (B) Proteolysis of RNase H by 200 µg/ml thermolysin. (C) Proteolysis of DHFR by 40 µg/ml subtilisin. Error bars show the standard errors from curve-fitting to a first-order rate equation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216078&req=5

pone-0111416-g001: Dependence of the apparent proteolysis rate constants (kp) on concentration of substrate proteins.(A) Proteolysis of DHFR by 80 µg/ml thermolysin. (B) Proteolysis of RNase H by 200 µg/ml thermolysin. (C) Proteolysis of DHFR by 40 µg/ml subtilisin. Error bars show the standard errors from curve-fitting to a first-order rate equation.
Mentions: To verify that our proteolysis condition satisfies pseudo-first-order kinetics, we determined the apparent first-order rate constants for proteolysis of DHFR by thermolysin at varying concentrations of DHFR (0.050–0.50 mg/mL) (Figure 1A). Interestingly, we observed that proteolysis of DHFR by thermolysin is slower with higher concentrations of DHFR. A ten-fold increase in DHFR concentration from 50 to 500 µg/ml resulted in a seven-fold decrease in the apparent rate constant. This dependence of the apparent kinetic constant on the substrate concentration suggests that the reaction does not satisfy the assumption of pseudo-first-order kinetics in which the apparent rate constant should depend only on the enzyme concentration. Fitting of the plot of remaining intact protein versus time to a first-order rate equation also shows non-random residuals, which indicates slight deviation from first-order kinetics (Figure S1).

Bottom Line: First-order kinetics has been assumed for the analysis of native-state proteolysis.By analyzing the kinetics of proteolysis with varying concentrations of substrate proteins and also with cleavage products, we found that the deviation from first-order kinetics results from product inhibition.A kinetic model including competitive product inhibition agrees well with the proteolysis time course and allows us to determine the uninhibited rate constant for proteolysis as well as the apparent inhibition constant.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, United States of America.

ABSTRACT
The proteolysis kinetics of intact proteins by nonspecific proteases provides valuable information on transient partial unfolding of proteins under native conditions. Native-state proteolysis is an approach to utilize the proteolysis kinetics to assess the energetics of partial unfolding in a quantitative manner. In native-state proteolysis, folded proteins are incubated with nonspecific proteases, and the rate of proteolysis is determined from the disappearance of the intact protein. We report here that proteolysis of intact proteins by nonspecific proteases, thermolysin and subtilisin deviates from first-order kinetics. First-order kinetics has been assumed for the analysis of native-state proteolysis. By analyzing the kinetics of proteolysis with varying concentrations of substrate proteins and also with cleavage products, we found that the deviation from first-order kinetics results from product inhibition. A kinetic model including competitive product inhibition agrees well with the proteolysis time course and allows us to determine the uninhibited rate constant for proteolysis as well as the apparent inhibition constant. Our finding suggests that the likelihood of product inhibition must be considered for quantitative assessment of proteolysis kinetics.

Show MeSH
Related in: MedlinePlus