Reassessing enzyme kinetics: Considering protease-as-substrate interactions in proteolytic networks

• Type of Material: Article
• Authors: Meghan C. Ferrall-Fairbanks, Georgia Institute of Technology; Chris A. Kieslich, Georgia Institute of Technology; Manu Platt, Emory University
• Language: English
• Date: 2020-02-11
• Publisher: National Academy of Sciences
• Publication Version: Final Published Version
• Copyright Statement: © 2020 National Academy of Science. All rights reserved.
• Final Published Version (URL): http://dx.doi.org/10.1073/pnas.1912207117
• Title of Journal or Parent Work: Proceedings of the National Academy of Sciences of the United States of America
• Volume: 117
• Issue: 6
• Start Page: 3307
• End Page: 3318
• Grant/Funding Information: This work was supported by NSF through the Science and Technology Center Emergent Behaviors of Integrated Cellular Systems Grant CBET-0939511 and, in whole or in part, by New Innovator Grant 1DP2OD007433-01 from the Office of the Director, NIH.
• Keywords: Cathepsin-k inhibitor; Cysteine cathepsins; Pycnodysostosis; Bone-mineral density; Functional expression; computational modeling; Postmenopausal women; Biochemical markers; Science & Technology - Other Topics; Multidisciplinary Sciences; Science & Technology; Osteoporosis; cysteine cathepsins; Osteoclasts; proteolysis; Abdominal aortic-aneurysms; extracellular matrix
• Research Categories: Health Sciences, Pathology; Chemistry, Biochemistry; Engineering, Biomedical

Abstract

Enzymes are catalysts in biochemical reactions that, by definition, increase rates of reactions without being altered or destroyed. However, when that enzyme is a protease, a subclass of enzymes that hydrolyze other proteins, and that protease is in a multiprotease system, protease-as-substrate dynamics must be included, challenging assumptions of enzyme inertness, shifting kinetic predictions of that system. Protease-on-protease inactivating hydrolysis can alter predicted protease concentrations used to determine pharmaceutical dosing strategies. Cysteine cathepsins are proteases capable of cathepsin cannibalism, where one cathepsin hydrolyzes another with substrate present, and misunderstanding of these dynamics may cause miscalculations of multiple proteases working in one proteolytic network of interactions occurring in a defined compartment. Once rates for individual protease-on-protease binding and catalysis are determined, proteolytic network dynamics can be explored using computational models of cooperative/competitive degradation by multiple proteases in one system, while simultaneously incorporating substrate cleavage. During parameter optimization, it was revealed that additional distraction reactions, where inactivated proteases become competitive inhibitors to remaining, active proteases, occurred, introducing another network reaction node. Taken together, improved predictions of substrate degradation in a multiple protease network were achieved after including reaction terms of autodigestion, inactivation, cannibalism, and distraction, altering kinetic considerations from other enzymatic systems, since enzyme can be lost to proteolytic degradation. We compiled and encoded these dynamics into an online platform (https://plattlab.shinyapps.io/catKLS/) for individual users to test hypotheses of specific perturbations to multiple cathepsins, substrates, and inhibitors, and predict shifts in proteolytic network reactions and system dynamics.