title: Cys-His proteases are among the wired proteins of the cell.
abstract: Integrated cell protein degradation can be paced by the transfer of reductive energy, as revealed by experimental agents of informative actions. The peptidolytic pair of Cys-His proteases can undergo oxidative reactions to inactive derivatives and inhibitory metal binding. Proton-dependent ionizations can modify ongoing activity. If the reaction rate of a Cys-His protease were found responsive to the ranges of metal/redox/proton factors regulated within the cell, then these factors might serve to link the peptidolytic reaction rate to cell controls. Here, cathepsin B (cat B) was found to be inhibited by Zn2+, Fe3+, and Cu2+ (1-50 microM) under excess GSH or DTT protease activators (6 mM). Under DTT or GSH (6 mM) the initial inhibitory action of Zn2+ is stable indefinitely; however, the inhibitory actions of Fe3+ and Cu2+ are reversed over approximately 1h. The 12-14 min half time of reversal of initial protease inhibition is correlated with the measured reduction of Fe3+ to Fe2+ by DTT or GSH (pH 5.5 or 6.5). Endogenous Fe2+ concentrations (100 microM) inhibit cat B only marginally. However, the inhibitory threshold of several microM Fe3+ is only a few percent oxidation of the endogenous pool. Without metals cat B reaction is reportedly proportional to GSH concentration, and is inhibited by increasing GSSG/GSH redox ratio. Following activation with GSH, cat B can be influenced by Fe3+/Fe2+, Cu2+/Cu+, and GSSG/GSH ratios and concentrations. Results are interpreted in relation to properties of the thiolate-imidazolium pair as illustrated by Dock modeling of their shared Fe3+ binding. It is proposed that the interaction of Cys-His with 1 electron transition between Fe2+ and Fe3+ serves as a sensor, signal integrator and switch wiring cat B reaction rate to the transfer of reductive energy in the presence of excess GSH. Speciated metals might also serve among electron acceptors transferring from reduced protease to oxygen. Results provide a model for pharmacologic redox switching of protease functions with metal-interactive drugs, and other nano-technology engineering.
authors.affiliation: Department of Pharmacology and Toxicology, School of Medicine, Wright State University, Cox Building, 3525 Southern Blvd, Kettering, OH 45429, USA. thomas.lockwood@wright.edu
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Lockwood
mesh_terms: D000818:Animals; D001665:Binding Sites; D001703:Biophysics / Q000379:methods*; D002401:Cathepsin B / Q000737:chemistry; D002417:Cattle; D003300:Copper / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D003553:Cystine / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D008247:Lysosomes / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry*; D011485:Protein Binding; D011522:Protons; D013154:Spleen / Q000378:metabolism; D013997:Time Factors; D015032:Zinc / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D007477:Ions; D011522:Protons; D003553:Cystine; D006639:Histidine; D003300:Copper; D007501:Iron; D010447:Peptide Hydrolases; D002401:Cathepsin B; D005978:Glutathione; D015032:Zinc; D003545:Cysteine; D010100:Oxygen; D004229:Dithiothreitol