Reconstructing the binding site of factor Xa in trypsin reveals ligand-induced structural plasticity.

In order to investigate issues of selectivity and specificity in protein-ligand interactions, we have undertaken the reconstruction of the binding pocket of human factor Xa in the structurally related rat trypsin by site-directed mutagenesis. Three sequential regions (the "99"-, the "175"- and the "190"- loops) were selected as representing the major structural differences between the ligand binding sites of the two enzymes. Wild-type rat trypsin and variants X99rT and X(99/175/190)rT were expressed in yeast, and analysed for their interaction with factor Xa and trypsin inhibitors.

S' subsite mapping of serine proteases based on fluorescence resonance energy transfer.

A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease.

Design and synthesis of fluorogenic trypsin peptide substrates based on resonance energy transfer.

An assay based on new internally quenched fluorogenic peptide substrates with the general structure 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)-Gly-Pro-Ala-Xaa-Leu-Ala-Ile-Gly-5-(2-aminoethylamino++ +)naphtha lene-1-sulfonic acid (EDANS), where Xaa = Arg, Lys, has been developed to measure proteolytic activity of trypsin and similar proteases. The kinetic parameters for the tryptic hydrolysis of DABCYL-Gly-Pro-Ala-Arg-Leu-Ala-Ile-Gly-EDANS are Km = 34 microM, kcat = 40 s-1, and kcat/Km = 1.17 x 10(6) M-1 s-1.

The specificity of prolyl endopeptidase from Flavobacterium meningoseptum: mapping the S' subsites by positional scanning via acyl transfer.

The S1'-S3' subsite specificity of prolyl endopeptidase from Flavobacterium meningoseptum was studied by acyl transfer to libraries of amino acid amides and peptides. Whereas the S1' and S3' subsites influence the specificity for the amino component by approximately one order of magnitude, the S2' subsite possesses a markedly higher specificity. Besides the high specificity for hydrophobic residues at P1'-P3', proline was efficiently bound by the S2' and S3' subsites of the enzyme.

Engineering the S1' subsite of trypsin: design of a protease which cleaves between dibasic residues.

The serine protease trypsin was converted into a site-specific protease which hydrolyzes peptides between dibasic residues. Trypsin exhibits a high S1 specificity for Arg and Lys residues. However, the S1' specificity of trypsin is very broad, with only a slight preference for hydrophobic residues in P1'.