Search

Ecotin, a homodimer protein of E. coli, is a unique member of canonical serine proteinase inhibitors, since it is a potent agent against a variety of serine proteinases having different substrate specificity. Monomers of ecotin are held together mostly by their long C-terminal strands that are arranged as a two-stranded antiparallel β-sheet in the functional dimer. One ecotin dimer can chelate two proteinase molecules, each of them bound to both subunits of ecotin at two different sites, namely the specific primary and the non-specific secondary binding sites. In this study the genes of wild type ecotin and its Met84 Arg P1 site mutant were truncated resulting in new forms of ecotin that lack 10 amino acid residues at their C-terminus. These mutants do not dimerize spontaneously, though in combination with trypsin they assemble into the familiar heterotetramer. Our data suggest that this heterotetramer exists even in extremely diluted solutions, and the interaction, which is responsible for the dimerization of ecotin, contributes to the stability of the heterotetrameric complex.

A new serine proteinase inhibitor, rapeseed trypsin inhibitor (RTI), has been isolated from rapeseed (Brassica napus var. oleifera) seed. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin with apparent dissociation constants of 3.0 x 10(-10) M and 4.1 x 10(-7) M, at pH 8.0 and 21 degrees C, respectively. The stoichiometry of both proteinase-inhibitor complexes is 1:1. The amino acid sequence of RTI consists of 60 amino acid residues, corresponding to an M(r) of about 6.7 kDa. The P1-P1' reactive site bond has been tentatively identified at position Arg20-Ile21. RTI shows no similarity to other serine proteinase inhibitors except the low molecular weight mustard trypsin inhibitor (MTI-2). RTI and MTI-2 could be members of a new class of plant serine proteinase inhibitors.

A new serine proteinase inhibitor, mustard trypsin inhibitor 2 (MTI-2), has been isolated from white mustard ( Sinapis alba L.) seed by affinity chromatography and reverse phase HPLC. The protein inhibits the catalytic activity of bovine β-trypsin and bovine α-chymotrypsin, with dissociation constants ( K d ) of 1.6 × 10 −10 M and 5.0 × 10 −7 M, respectively, at pH 8.0 and 21°C, the stiochiometry of both proteinase-inhibitor complexes being 1:1. The amino acid sequence of MTI-2, which was determined following S -pyridylethylation, is comprised of 63 residues, corresponding to a molecular weight of about 7 kDa, and shows only extremely limited homology to other serine proteinase inhibitors.

Thermitase is a thermostable member of the subtilisin family of serine proteases. Four independently determined crystal structures of the enzyme are compared in this study: a high resolution native one and three medium resolution complexes of thermitase with eglin-c, grown from three different calcium concentrations. It appeared that the B-factors of the thermitase eglin complex obtained at 100 mM CaCl2 and elucidated at 2.0 A resolution are remarkably similar to those of the 1.4 A native structure: the main chain atoms have an rms difference of only 2.3 A 2; for all atoms this difference is 4.6 A 2. The rms positional differences between these two structures of thermitase are 0.31 A for the main chain atoms and 0.58 A for all atoms. There results show that not only atomic positions but also temperature factors can agree well in X-ray structures determined entirely independently by procedures which differ in virtually every possible technical aspect.A detailed comparison focussed on the effects of eglin binding on the structure of thermitase. Thermitase can be considered as consisting of (1) a central core of 94 residues, plus (2) four segments of 72 residues in total which shift as rigid bodies with respect to the core, plus (3) the remaining 113 residues which show small changes but, however, cannot be described as rigid bodies. The central cores of native thermitase and the 100 mM CaCl2 thermitase:eglin complex have an rims deviation of 0.13 A for 376 main chain atoms. One of the segments, formed by loops of the strong calcium binding site, shows differences up to 1.0 A in C-alpha positions. These are probably due to crystal packing effects.The three other segments, comprising 51 residues, are affected conformational changes upon eglin binding so that the P1 to P3 binding pockets of thermitase broaden by 0.4 to 0.7 A. The residues involved in these changes correspond with residues which change position upon inhibitor binding in other subtilisins. This suggests that an induced fit mechanism is operational during substrate recognition by subtilisins.