Hydrolases with Medical Relevance: Proteases and Their Inhibitors in Acute Pancreatitis and Glycoside Hydrolases for the Creation of Universal Blood

In this dissertation, the roles of several medically relevant hydrolases, focusing on proteases involved in AP and one of their inhibitors, as well as glycoside hydrolases used for the creation of universal blood, were successfully investigated. The cysteine proteases CTSB and CTSL were expressed in soluble and active form using E. coli SHuffle® T7 Express cells, achieving high expression yields of 80 ± 2 mg L-1 for CTSB_N-6xHis and 37 ± 2 mg L-1 for CTSL_N-6xHis using TB medium (Article I). After recombinant expression as proenzymes, the enzymes were purified by immobilized nickel ion-affinity chromatography, activated in vitro, and characterized. Using these recombinantly expressed enzymes, their role and the importance of the endogenous inhibitor CST3 in terms of the onset of AP were investigated by a structure-function-analysis, including biochemical analyses, site-directed mutagenesis, and molecular dynamic simulations in combination with an experimental disease model of AP using CST3 deficient mice (Article II). We could show that CST3 is a key regulator of CTSB and CTSL activity during the development of AP. We found that CST3 can be cleaved at the two cleavage sites K24 and R28 by trypsin disabling the inhibition of the trypsinogen activating enzyme, CTSB, but not the trypsin(ogen) inactivating enzyme, CTSL. Interestingly, we discovered that dimerized CST3 even enhances CTSB activity 3-fold by binding to a newly discovered allosteric pocket. The presence of the allosteric binding pocket was confirmed using activity tests with a mutant where six residues of the allosteric pocket of CTSB were deleted by site-directed mutagenesis. Thereby, CST3 shifts from a CTSB inhibitor to an activator, which fuels the intrapancreatic protease cascade during AP onset. In the second part of this thesis, previously unstudied B antigen removing enzymes of the GH110 family were successfully investigated (Article III). We found that PpaGal is most efficient in removing B antigen from the surface of RBCs and its activity was ...