TAILIEUCHUNG - Báo cáo khoa học: N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity

Yeast IA3 aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, sac-charopepsin. Even aspartic proteinases that are very closely similar to saccharopepsin (. the vacuolar enzyme fromPichia pastoris) are not sus-ceptible to significant inhibition. | ỊFEBS Journal N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity Tim J. Winterburn1 Lowri H. Phylip1 Daniel Bur2 David M. Wyatt1 Colin Berry1 and John Kay1 1 Schoolof Biosciences Cardiff University UK 2 Actelion Pharmaceuticals Ltd Allschwil Switzerland Keywords aspartic proteinase inhibition IA3 inhibitor engineering Pichia aspartic proteinase specificity relaxation Correspondence J. Kay School of Biosciences Cardiff University Museum Avenue Cardiff CF10 3US UK Fax 44 029 20 87 41 16 Tel 44 029 20 87 41 24 E-mail kayj@ Received 30 March 2007 revised 23 May 2007 accepted 25 May 2007 doi Yeast IA3 aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme sac-charopepsin. Even aspartic proteinases that are very closely similar to saccharopepsin . the vacuolar enzyme from Pichia pastoris are not susceptible to significant inhibition. The Pichia proteinase was selected as the target for initial attempts to engineer IA3 to re-design the specificity. The IA3 polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence. Alterations made by deletion or exchange of the residues in the C-terminal segment of these polypeptides had only minor effects. By contrast extension of each of these wild-type and chimaer-ic polypeptides at its N-terminus by an MK H 7MQ sequence generated inhibitors that displayed subnanomolar potency towards the Pichia enzyme. This gain-in-function was completely reversed upon removal of the extension sequence by exopeptidase trimming. Capture of the potentially positively charged aromatic histidine residues of the extension by remote negatively charged side-chains which were identified in the Pichia enzyme by modelling may increase the local IA3 concentration and create an anchor that enables the N-terminal segment residues to be .

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