title: Characterization of potato proteinase inhibitor II reactive site mutants.
abstract: Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (Ki = 0.4 nM) and chymotrypsin (Ki = 0.9 nM), and is quite robust towards mutations at positions P2 or P1'. In contrast, the first reactive site inhibits only chymotrypsin (Ki = 2 nM), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.
authors.affiliation: Department of Molecular Biology, Center for Plant Breeding and Reproduction Research (CPRO), Wageningen, the Netherlands. m.j.beekwilder@plant.wag-ur.nl
authors.forename: J
authors.initials: J
authors.lastname: Beekwilder
authors.forename: B
authors.initials: B
authors.lastname: Schipper
authors.forename: P
authors.initials: P
authors.lastname: Bakker
authors.forename: D
authors.initials: D
authors.lastname: Bosch
authors.forename: M
authors.initials: M
authors.lastname: Jongsma
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011480:Protease Inhibitors; C034104:proteinase inhibitor II protein, plant; D004247:DNA; D014357:Trypsin