Heterologous expression of three plant serpins with distinct inhibitory specificities.
For the first time, inhibitory plant serpins, including WSZ1 from wheat, BSZ4, and the previously unknown protein BSZx from barley, have been expressed in Escherichia coli, and a procedure for fast purification of native plant serpins has been developed. BSZx, BSZ4, and WSZ1 were assayed for inhibitory activity against trypsin, chymotrypsin, and cathepsin G, and cleavage sites in the reactive center loop were identified by sequencing. BSZx proved to be a potent inhibitor with specific, overlapping reactive centers either at P1 Arg for trypsin or at P2 Leu for chymotrypsin. At 22 ;C, the apparent rate constant for chymotrypsin inhibition at P2 (ka = 9.4 x 10(5) M-1 s-1) was only four times lower than for trypsin at P1 (ka = 3.9 x 10(6) M-1 s-1), and the apparent inhibition stoichiometries were close to 1. Furthermore, our data suggest that cathepsin G was inhibited by BSZx (ka = 3.9 x 10(6) M-1 s-1) at both the P1 Arg and P2 Leu. These results indicate a unique adaptability of the reactive center loop of BSZx. WSZ1 inhibited chymotrypsin (ka = 1.1 x 10(5) M-1 s-1) and cathepsin G (ka = 7.6 x 10(3) M-1 s-1) at P1 Gln and not, as for BSZx, at the more favorable P2 Leu. BSZ4 inhibited cathepsin G (ka = 2.7 x 10(4) M-1 s-1) at P1 Met but was hydrolyzed by trypsin and chymotrypsin. The three plant serpins formed stable SDS-resistant complexes with the proteinases in accordance with the kinetic data.
Dahl
S W
SW
Department of Biochemistry and Nutrition, Technical University of Denmark, DK-2800 Lyngby, Denmark.
Rasmussen
S K
SK
Hejgaard
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D001467:Hordeum / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012697:Serine Endopeptidases; D015843:Serpins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D014908:Triticum / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
C101795:BSZx protein, Hordeum vulgare; D017931:DNA Primers; C101794:PAZ1 protein, Hordeum vulgare; D010940:Plant Proteins; D011994:Recombinant Proteins; D015843:Serpins; C099007:WSZ1 protein, Triticum aestivum; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D056649:Cathepsin G; D014357:Trypsin