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.
Highlights
Native-state proteolysis is a useful experimental approach to investigate transient partial unfolding in proteins under native conditions [1,2,3,4]
We examined the proteolysis kinetics of two intact proteins E. coli dihydrofolate reductase (DHFR) and E. coli ribonuclease HI (RNase H) by two nonspecific proteases, thermolysin and subtilisin under various conditions
The apparent proteolysis rate constant is dependent on protein substrate concentration When substrate concentration is low, the reaction catalyzed by an enzyme has pseudo-first-order kinetics, which can be described by a single exponential decay with an apparent first-order rate constant determined by Vmax/Km or the product of kcat/Km and the enzyme concentration
Summary
Native-state proteolysis is a useful experimental approach to investigate transient partial unfolding in proteins under native conditions [1,2,3,4]. This method exploits the common observation that proteolysis of folded proteins requires partial unfolding. By determining kp experimentally and approximating kint from proteolysis kinetics of an unstructured peptide substrate by the same protease, one can determine Kop. To avoid the bias from the sequence specificity of proteases in probing partial unfolding, we employ nonspecific proteases (e.g. thermolysin) in native-state proteolysis
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