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N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity

Winterburn, Timothy John, Phylip, Lowri Haf, Bur, Daniel, Wyatt, David Michael, Berry, Colin and Kay, John 2007. N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity. FEBS journal 274 (14) , pp. 3685-3694. 10.1111/j.1742-4658.2007.05901.x

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Abstract

Yeast IA3 aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, saccharopepsin. Even aspartic proteinases that are very closely similar to saccharopepsin (e.g. 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 chimaeric 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 harboured in closer proximity to the enzyme active site, thus promoting their interaction. In saccharopepsin, some of the counterpart residues are different and, consistent with this, the N-terminal extension of each IA3 polypeptide was without major effect on the potency of interaction with saccharopepsin. In this way, it is possible to convert IA3 polypeptides that display little affinity for the Pichia enzyme into potent inhibitors of this proteinase and thus broaden the target selectivity of this remarkable small protein.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Uncontrolled Keywords: aspartic proteinase inhibition ; IA3 ; inhibitor engineering ; Pichia aspartic proteinase ; specificity relaxation
ISSN: 1742-464X
Last Modified: 04 Jun 2017 01:38
URI: http://orca.cf.ac.uk/id/eprint/1111

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