Understanding cellular house-keeping of proteins by shedding light on the reaction mechanism displayed by prolidase enzymes
Issues of importance for the application: Per-Olof Syrén is Associate Professor at KTH. This project is funded by a VR grant (dnr 2013-5138), by the Swedish Foundation for Strategic Research (SSF) and FORMAS through a 2016 Future Research Leader grant. My research was evaluated by VR to show “great potential for human kind”. Published papers using PDC #2016/1-34: 6 (2 in impact factor >5). The present investigation is centered on deciphering the reaction mechanism displayed by prolidase enzymes, which is poorly understood at present. By using state of the art quantum mechanical calculations, we will elucidate the details of catalysis at the molecular level for these bi-metal proteases with unique capabilities of degrading tertiary amide bonds that are introduced in proteins and peptides throughout all kingdoms of life to mediate protein stability and bioactivity. The results from the project are thus expected to be of paramount importance in medicine, as well as in industrial biotechnology. Great progress was made in SNIC 2016/1-34 as three transition states for this complex biocatalysis was successfully unraveled. However, important mechanistic details are still missing, including key transition state structures, which will be addressed in the present investigation. Cells use the incorporation of proline amino acids as a general strategy to protect peptides from degradation by proteolytic enzymes. Hence, the tertiary amide bond is crucial for the housekeeping of cellular proteins, which is of high importance in cancer and other diseases. Interestingly, the assembly of the HIV-virus requires the hydrolysis of a tertiary amide bond. Moreover, natural products containing prolines - or other tertiary reacting nitrogen atoms – display potent bioactivities and are key players in cell signaling. One important example is the blood-pressure-controlling peptide bradykinin. Hence, a detailed understanding of the reaction mechanism by which these peptides are metabolized by prolidase enzymes would be desirable and of high importance in medicine and biochemistry. Proteases and amidases are ubiquitous enzymes with important functions in the metabolism, immune response, protein and viral maturation, apoptosis, regeneration and signal transduction. The crucial importance of proteases for homeostasis is illustrated by the fact that many genetic diseases are closely associated with proteolytic activity. Moreover cascades of proteases are key factors for cancer and tumor progression and Alzeimer’s disease. It is therefore of significant importance to increase our understanding of the catalytic mechanism displayed by these enzymes and to elucidate the details of catalysis at the molecular level, since this would aid in drug design. We have previously shown that proteases display two distinct catalytic strategies to facilitate proteolysis (e.g. see the PDC-supported work Chem. Commun. 2015, 51, 17221). Our work has furthermore received a positive response from the Scientific community. It is envisaged that the present investigation centered on substrates with tertiary amide bonds will reveal additional catalytic strategies. The results generated will moreover be of high value in industrial biotechnology, where tertiary amide bonds are key motifs in biopharmaceuticals and in novel bio-based nanomaterials.