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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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Related in: MedlinePlus

Determination of k0 and Ki by global fitting.(A) Fractions of intact protein remaining after proteolysis of 50 (▪), 100 (△), 200 (▾), 300 (○), and 500 (•) µg/ml DHFR by 80 µg/ml thermolysin are shown with the global fitting by Eq. 2. (B) Residuals are shown for fitting proteolysis of 50 µg/ml (•) or 500 µg/ml (○) DHFR by a first-order rate equation. Residuals are shown for fitting the proteolysis time courses predicted by global fitting parameters (solid lines in A) for 50 µg/ml (―) or 500 µg/ml (---) DHFR to a first-order rate equation.
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pone-0111416-g003: Determination of k0 and Ki by global fitting.(A) Fractions of intact protein remaining after proteolysis of 50 (▪), 100 (△), 200 (▾), 300 (○), and 500 (•) µg/ml DHFR by 80 µg/ml thermolysin are shown with the global fitting by Eq. 2. (B) Residuals are shown for fitting proteolysis of 50 µg/ml (•) or 500 µg/ml (○) DHFR by a first-order rate equation. Residuals are shown for fitting the proteolysis time courses predicted by global fitting parameters (solid lines in A) for 50 µg/ml (―) or 500 µg/ml (---) DHFR to a first-order rate equation.

Mentions: Because the reaction products are clearly the inhibitory agent, we fit time courses of DHFR proteolysis (Figure 3A) using a competitive inhibition model:(2)


Product inhibition in native-state proteolysis.

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

Determination of k0 and Ki by global fitting.(A) Fractions of intact protein remaining after proteolysis of 50 (▪), 100 (△), 200 (▾), 300 (○), and 500 (•) µg/ml DHFR by 80 µg/ml thermolysin are shown with the global fitting by Eq. 2. (B) Residuals are shown for fitting proteolysis of 50 µg/ml (•) or 500 µg/ml (○) DHFR by a first-order rate equation. Residuals are shown for fitting the proteolysis time courses predicted by global fitting parameters (solid lines in A) for 50 µg/ml (―) or 500 µg/ml (---) DHFR to a first-order rate equation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111416-g003: Determination of k0 and Ki by global fitting.(A) Fractions of intact protein remaining after proteolysis of 50 (▪), 100 (△), 200 (▾), 300 (○), and 500 (•) µg/ml DHFR by 80 µg/ml thermolysin are shown with the global fitting by Eq. 2. (B) Residuals are shown for fitting proteolysis of 50 µg/ml (•) or 500 µg/ml (○) DHFR by a first-order rate equation. Residuals are shown for fitting the proteolysis time courses predicted by global fitting parameters (solid lines in A) for 50 µg/ml (―) or 500 µg/ml (---) DHFR to a first-order rate equation.
Mentions: Because the reaction products are clearly the inhibitory agent, we fit time courses of DHFR proteolysis (Figure 3A) using a competitive inhibition model:(2)

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