tailieunhanh - Báo cáo khoa học: Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate

The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a serralysin-like metzincin from the entomo-pathogenic microorganism,Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin andb-lipotropin, and of 15 synthetic peptides, was investigated. | ễFEBS Journal Cleavage site analysis of a serralysin-like protease PrtA from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate Judit Marokhazi1 Nikolett Mihala2 Ferenc Hudecz2 3 Andras Fodor1 László Graf1 4 and Istvan Venekei1 1 Department of Biochemistry Eotvos Lorand University Budapest Hungary 2 Department of Organic Chemistry Eotvos Lorand University Budapest Hungary 3 Research Group of Peptide Chemistry Hungarian Academy of Sciences Budapest Hungary 4 Biotechnology Research Group Hungarian Academy of Sciences Budapest Hungary Keywords cleavage site serralysin specific substrate metalloprotease PrtA of Photorhabdus Correspondence I. Venekei Department of Biochemistry Eotvos Lorand University Budapest Pazmany Peter setany 1 C. 1117 Hungary Fax 36 1 381 2172 Tel 36 1 209 0555 8777 E-mail venekei@ Received 5 December 2006 revised 9 February 2007 accepted 12 February 2007 doi The aim of this study was the development of a sensitive and specific substrate for protease A PrtA a serralysin-like metzincin from the entomo-pathogenic microorganism Photorhabdus. First cleavage of three biological peptides the A and B chains of insulin and b-lipotropin and of 15 synthetic peptides was investigated. In the biological peptides a preference for the hydrophobic residues Ala Leu and Val was observed at three substrate positions P2 P1 and P2 . At these positions in the synthetic peptides the preferred residues were Val Ala and Val respectively. They contributed to the efficiency of hydrolysis in the order P1 P2 P2 . Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide a fluorescence-quenched substrate N- 4- 4 dimethylamino phenylazo benzoyl-EVYAVES-5- .