A new series of peptide inhibitors of human Factor VIIa (FVIIa) has been identified and affinity matured from naive and partially randomized peptide phage libraries selected against the immobilized tissue factor x Factor VIIa (TF x FVIIa) complex. These "A-series" peptides contain a single disulfide bond and a 13-residue minimal core required for maximal affinity. They are exemplified by peptide A-183 (EEWEVLCWTWETCER), which binds at a newly identified exosite on the FVIIa protease domain, described in the accompanying report [Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) Biochemistry 40, 9522-9531]. A-183 was obtained from a trypsin digest of A-100-Z, a recombinant protein comprising A-183 and the Z domain of protein A. Surprisingly, A-183 was a very potent inhibitor of TF x FVIIa, inhibiting activation of Factor X (FX) and Factor IX and amidolytic activity of Chromozym t-PA with IC50 values of 1.6 +/- 1.2, 3.5 +/- 0.3, and 8.5 +/- 3.5 nM, respectively. Kinetic analysis revealed that A-183 was a partial (hyperbolic) mixed-type inhibitor of FX activation having a Ki of 200 pM as well as a partial competitive inhibitor of amidolytic activity. The A-series peptides were also specific and potent inhibitors of TF-dependent clotting as measured in a prothrombin time (PT) clotting assay and had no effect on the TF-independent activated partial thromboplastin time. At saturating concentrations of peptide, the maximal extent by which A-183 and A-100-Z inhibited the rate of FX activation was 78 +/- 3 and 89 +/- 6%, respectively. The degree of inhibition of the rate of FX activation correlated with a maximum fold prolongation in the PT assay of 1.8-fold for A- 183 and 3.3-fold for A-100-Z. The A-series peptides represent a new class of peptide exosite inhibitors that are capable of attenuating, rather than completely inhibiting, the activity of TF x FVIIa, potentially leading to anticoagulants with an increased therapeutic window.

A new inhibitory peptide binding exosite on the protease domain of coagulation Factor VIIa (FVIIa) has been identified. A novel series of peptide inhibitors of FVIIa, termed the "A-series" peptides, identified from peptide phage libraries and exemplified by peptide A-183 [Dennis, M. S., Roberge, M., Quan, C., and Lazarus, R. A. (2001) Biochemistry 40, 9513-9521], specifically bind at a site that is distinct from both the active site and the exosite of another recently described peptide inhibitor of FVIIa, E-76 [Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., and Lazarus, R. A. (2000) Nature 404, 465-4701. Peptide A-183 prolonged TF-dependent clotting in human, but not rabbit plasma. Thus, a panel of human FVIIa mutants, containing 70 of the 76 rabbit sequence differences in the protease domain, localized the binding site to residues in the 60s loop and the C-terminus. The location of the exosite was refined by a series of FVIIa alanine mutants, which showed that proximal residues Trp 61 and Leu 251 were critical for binding. Kinetic and equilibrium binding constants for zymogen FVII, FVIIa and TF x FVIIa were determined using immobilized N-terminal biotinylated A-183 by surface plasmon resonance. No peptide binding to nine other human serine proteases was observed. Key residues on the peptide were determined from binding to FVIIa and inhibition of FX activation using a series of alanine mutants of A-183 fused to the Z domain of protein A. Analysis of the mutagenesis data is presented in the context of a crystal structure of A-183 in complex with a version of zymogen FVII [Eigenbrot, C., Kirchhofer, D., Dennis, M. S., Santell, L., Lazarus, R. A., Stamos, J., and Ultsch, M. H. (2001) Structure 9, 627-636]. The shape and proximity of this exosite to the active site may lend itself towards the design of new anticoagulants that inhibit FVIIa.

The substrate-binding domains of six glycanases from Streptomyces lividans were investigated to determine their specificity towards cellulose and xylan. Based upon amino acid sequence similarities, four of the six domains could be assigned to existing cellulose-binding domain families. However, the binding domains of xylanase A and arabinofuranosidase B could not be classified in any of the known families and should therefore be classified as members of a new family. Evidence is also presented that this new family is one of true xylan-binding domains.

The role of four aromatic residues (W85, Y172, W266 and W274) in the structure-function relationship in xylanase A from Streptomyces lividans (XlnA) was investigated by site-directed mutagenesis where each residue was subjected to three substitutions (W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four amino acids are highly conserved among family 10 xylanases and structural data have implicated them in substrate binding at the active site. Far-UV circular dichroism spectroscopy was used to show that the overall structure of XlnA was not affected by any of these mutations. High-performance liquid chromatographic analysis of the hydrolysis products of birchwood xylan and xylopentaose showed that mutation of these aromatic residues did not alter the enzyme's mode of action. As expected, though, it did reduce the affinity of XlnA for birchwood xylan. A comparison of the kinetic parameters of different mutants at the same position demonstrated the importance of the aromatic nature of W85, Y172 and W274 in substrate binding. Replacement of these residues by a phenylalanine resulted in mutant proteins with a K(M) closer to that of the wild-type protein in comparison with the other mutations analyzed. The kinetic analysis of the mutant proteins at position W266 indicated that this amino acid is important for both substrate binding and efficient catalysis by XlnA. These studies also demonstrated the crucial role of these active site aromatic residues for the thermal stability of XlnA.

On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.