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observed for few residues including Arg, Har, Gnf and for Lys. No significant proteolysiswas detected for Orn, Dab and Amf. In position P 3 human furin was able to hydrolyzedalmost all residues except Dab. The highest fluorescent intensity was recorded for Arg andLys. A slightly lower response was observed for Gnf. Substrate preferences in position P 2was similar as in position P 4 . Arg and Lys followed by Gnf and Har were hydrolyzed withthe highest rate. Low fluorescence was observed for the remaining amino acids. Allpeptides that undergoes proteolysis were characterized and their kinetic parameters(k cat and K M ) are presented in Table 1.Based on the selected substrate ABZ-Gnf-Gnf- Lys-Arg-ANB-NH 2 , a set of aldehydeinhibitors were designed. FRET substrates were converted into corresponding peptidealdehydes. Simply, ANB-NH 2 present at the C-terminus of peptide was replaced byaldehyde group yielding the peptide aldehyde. The solid support was used to synthesizeTable 1. Physicochemical characteristic and kinetic parameters of human furin substratesSubstratet R[min]MW calc.(determ)k cat[s -1 ]K M[μM]k cat / K M[s -1 ×M -1 ]ABZ-Gnf-Gnf-βLys-Arg-ANB-NH 2 12.8ABZ-Gnf-Gnf-Har-Arg-ANB-NH 2 10.2ABZ-Gnf-Gnf-Gnf-Arg-ANB-NH 2 14.3ABZ-Gnf-Gnf-Arg-Arg-ANB-NH 2 9.81007.1(1007.3)7.8±0.4 12.5±1.2 645.4×10 31028.1(1028.4)4.2±0.2 39.4±2.7 106.7×10 31069.2(1069.3)4.9±0.4 11.8±2.1 415.1×10 31014.3(1014.3)7.2±0.8 15.4±1.3 467.2×10 3such peptide derivative. A set of peptide aldehydes (ABZ-Gnf-Gnf- Lys-X-H) modified inposition X by amino acid residues used for library construction (Figure 1) was synthesized.The preliminary activity test of obtained compounds indicate that the activity of humanfurin is diminished when incubated with ABZ-Gnf-Gnf- Lys-Gnf-H, and ABZ-Gnf-Gnf-Lys-Har-H. Peptide aldehydes with Arg, D-Arg, Orn and Amf displayed lower inhibitora potency. This animal The lowest did not PC like inhibition it was observed for Lys, Dab and Ala.In summary we would like to conclude that human furin is able to recognize andhydrolyze peptide with non proteinogenic amino acid within its sequence. Among them thehighest value of the specificity constant was obtained for ABZ-Gnf-Gnf- Lys-Arg-ANB-NH 2 (k cat /K M = 645×10 3 M -1 ×s -1 ).The peptide aldehydes that sequence was based onselected FRET substrate were able to inhibit human furin activity with values of inhibitonconstants at nanomolar range.AcknowledgmentsThis work was supported by Polish Ministry of Science and Higher Education under grant1600/B/H03/2009/36.References1. Mollay, S.S., Bresnhahan, P.A., Leppla, S.H., Klimpel, K.R., Thomas, G. J. Biol. Chem. 267,16396-16402 (1992).2. Steiner, D.F. Curr. Opin. Chem. Biol. 2, 31-39 (1998).3. Wysocka, M., Kwiatkowska, B., Rzadkiewicz, M., Lesner, A., Rolka, K. Comb. Chem. HighThrough S. 3, 171-180 (2007).4. Furka, A., Sebestyn, F., Asgedom, M., Dib, G. Int. J. Peptide Prot. Res. 37, 487-494 (1991).199