title: Two reactive site locations and structure-function study of the arrowhead proteinase inhibitors, A and B, using mutagenesis.
abstract: The arrowhead (Sagittaria sagittifolia, Linn.) proteinase inhibitor A and B are double-headed and multifunctional, consisting of 179 amino acid residues with three disulfide bridges. Both their primary structures and cDNA sequences have been elucidated [Yang, H. L., Luo, R. S., Wang, L. X., Zhu, D. X., & Chi, C. W. (1992) J. Biochem. 111, 537; Xu, W. F., Tao, W. K., Gong, Z. Z., & Chi, C. W. (1993) J. Biochem. 113, 153; Luo, M. J., Lu, W. Y., & Chi, C. W. (1997) J. Biochem. (in press)]. Though they share 91% homology, they are different in inhibitory activities. Sequence analysis of their full-length cDNAs showed that there are seven extra residues in the C-terminal part which might be cleaved off by proteinase post-processing. To locate the reactive sites and study the structure-function relationship of the two forms A and B, the genes coding for the mature inhibitor B and its extended form were respectively cloned into the secretion expression vector, pVT102U/alpha, and expressed in Saccharomyces cerevisiae strain S-78. Both of the gene products were purified and characterized to have the same inhibitory activities as the natural one. The gene product of the extended form was a mixture with the extended C-terminal part of the inhibitor either completely or partially removed. The two previously predicted reactive site residues, Lys-44 and Arg-76 of inhibitor B, were then respectively substituted with Ala by site-directed mutagenesis and expressed. As compared with the natural inhibitor, each of the mutants could only inhibit one molecule, instead of two molecules of trypsin, and displayed an inhibitory activity against elastase, thus confirming the location of the two reactive sites in the inhibitors. The gene coding for inhibitor A, which for some reason could not be expressed in S. cereviciae, was successfully expressed in the reconstructed plasmid pET-1522bx in Escherichia coli strain BL21 with the expressed product existing in the inclusion body. After denaturation and renaturation, the active inhibitor A was obtained and purified by anhydrotrypsin affinity chromatography. Using site-directed mutagenesis, two residues of inhibitor A, namely, Ser-82 and Leu-87, prominently different from Leu-82 and Arg-87 in inhibitor B, were replaced by these two corresponding residues, respectively. As compared with the natural inhibitor A, its S82L mutant showed a lower inhibitory activity toward trypsin, whereas a higher activity was found in the L87R mutant. Meanwhile, both of their chymotrypsin inhibitory activities became weaker than the natural one. The important accessary role of the residue of position 87 in causing the difference in inhibitory properties between inhibitor A and B was discussed.
authors.affiliation: State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, China.
authors.forename: Z W
authors.initials: ZW
authors.lastname: Xie
authors.forename: M J
authors.initials: MJ
authors.lastname: Luo
authors.forename: W F
authors.initials: WF
authors.lastname: Xu
authors.forename: C W
authors.initials: CW
authors.lastname: Chi
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: C080655:API-A protein, Sagittaria sagittifolia; D010940:Plant Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C047493:proteinase inhibitor B, arrowhead root