title: Product inhibition in native-state proteolysis.
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.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, United States of America.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Kasper
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, United States of America.
authors.forename: Elizabeth C
authors.initials: EC
authors.lastname: Andrews
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, United States of America.
authors.forename: Chiwook
authors.initials: C
authors.lastname: Park
mesh_terms: D007700:Kinetics; D008954:Models, Biological; D059748:Proteolysis; D016914:Ribonuclease H / Q000378:metabolism; D020860:Subtilisin / Q000037:antagonists & inhibitors / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013820:Thermolysin / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D013762:Tetrahydrofolate Dehydrogenase; D016914:Ribonuclease H; D020860:Subtilisin; D013820:Thermolysin