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Volume 271, Number 41, Issue of October 11, 1996 pp. 25083-25088
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Heterologous Expression of Three Plant Serpins with Distinct Inhibitory Specificities

(Received for publication, April 5, 1996)

Søren W. Dahl ^§ , Søren K. Rasmussen ^§ and Jørn Hejgaard

From the  Department of Biochemistry and Nutrition, Technical University of Denmark, DK-2800 Lyngby, Denmark and ^§ Plant Genetics, Environmental Science and Technology Department, Risø National Laboratory, DK-4000 Roskilde, Denmark

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 (k_a = 9.4 × 10⁵ M¹ s¹) was only four times lower than for trypsin at P₁ (k_a = 3.9 × 10⁶ M¹ s¹), and the apparent inhibition stoichiometries were close to 1. Furthermore, our data suggest that cathepsin G was inhibited by BSZx (k_a = 3.9 × 10⁶ M¹ s¹) at both the P₁ Arg and P₂ Leu. These results indicate a unique adaptability of the reactive center loop of BSZx. WSZ1 inhibited chymotrypsin (k_a = 1.1 × 10⁵ M¹ s¹) and cathepsin G (k_a = 7.6 × 10³ M¹ s¹) at P₁ Gln and not, as for BSZx, at the more favorable P₂ Leu. BSZ4 inhibited cathepsin G (k_a = 2.7 × 10⁴ M¹ s¹) 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.

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