Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (k(a) ') for irreversible complex formation with pancreas serine proteinases at 24degreesC were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P-1 -P-1 ' Thrdown arrowSer, were efficient inhibitors of pancreas elastase (k(a) ' > 10(5) M (-1) s(-1) ). Only OSZb was also an inhibitor of chymotrypsin at the same site (k(a) ' = 0.9 x 10(5) M (-1) s(-1) ). OSZc was a fast inhibitor of trypsin at P-1 -P-1 ' Argdown arrowSer (k(a) ' = 4 x 10(6) M (-1) s(-1) ); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant k(d) = 1.4 x 10(-4) s(-1) . OSZc was also an inhibitor of chymotrypsin (k(a) ' > 10(6) M (-1) s(-1) ), presumably at the overlapping site P-2 -P-1 Aladown arrowArg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P-1 -P-1 ' Tyrdown arrowSer, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.