| Carboxypeptidase M (EC 3.4.17.12) (CPM) belongs to the family of the carboxypeptidases. Compared to other basic CPs such as CPD and CPE the information on the substrate preference of CPM, CPU and CPN beyond P1' (Arg/Lys) was rather limited. The aim of this thesis was therefore a more extended study of the substrate specificity of the membrane-bound CPM for synthetic substrates in order to contribute to the development of potent and selective inhibitors. In addition, we hoped that this knowledge would contribute to the understanding of the preference of the enzyme for human substrates and the elucidation of a physiological role for CPM. Despite the fact that the function(s) of CPM is not fully understood several suggestions have been made since its discovery more then two decades ago. Based on potential substrates and its presence, often on the boundary between the host and environment, a role in inflammation was proposed.;We set up a recombinant system for the production of CPM in sufficient amounts to realize this project and purified the enzyme from natural and recombinant source. Interestingly, we noted that not only the ultimate (P1') but also the penultimate (P1) and adjacent P2 residue dictated the substrate specificity kcat and KM of CPM. Our results indicate that P1-S1 interactions play a role in substrate binding. Extending the substrate from P3 up to P7 had little effect on the catalytic parameters of CPM.;The next logical step towards unraveling the substrate preference of CPM was the in vitro study of the breakdown of some endogenous peptides (C3a, CCL1 and SDF-1alpha). Since confusion existed concerning the role of Tyr248 (CPA) in the catalytic process we opted to mutate the analogous amino acid in CPM.;A last objective was the exploration of the physiological role for CPM. The truncation of the C-terminal Lys of SDF-1alpha by CPM decreased the migration of cells in vitro and this effect could not be attributed to a less efficient binding of the chemokine to the CXCR4 receptor. The inhibition of CP activity of the cells in vitro increased the chemotaxis suggesting a role for a CP. |
