Cys-His proteases are among the wired proteins of the cell.
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.
Lockwood
Thomas D
TD
Department of Pharmacology and Toxicology, School of Medicine, Wright State University, Cox Building, 3525 Southern Blvd, Kettering, OH 45429, USA. thomas.lockwood@wright.edu
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
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