CHARACTERIZATION OF THE P-2' AND P-3' SPECIFICITIES OF THROMBIN USINGFLUORESCENCE-QUENCHED SUBSTRATES AND MAPPING OF THE SUBSITES BY MUTAGENESIS

The importance of substrate residues P-2' and P-3' On thrombin catalys is has been investigated by comparing the hydrolysis of a series of fl uorescence-quenched substrates, Each consisted of a 10-residue peptide , carrying a 2-aminobenzoyl (Abz) group at the N-terminus, and a penul timate 2,4-dinitrophenyl (Dnp) derivatized lysine. Cleavage of such a peptide relieves the intramolecularly-quenched fluorescence, allowing determination of the kinetic parameters. The nature of the P-2' residu e was found to have a major influence on the rate of cleavage: the k(c at)/K-m value for the hydrolysis of the Arg-Ser bond in bz-Val-Gly-Pro -Arg-Ser-Phe-Leu-Leu-Lys(Dnp)-Asp-OH was nearly 3 orders of magnitude higher than that for the hydrolysis of the same substrate with asparta te instead of phenylalanine at the P-2' position. Comparatively, the P -3' Side chain was less important: the k(cat)/K-m value for the substr ate with the least effective residue (aspartate) was only 33 times low er than that of the substrate with the most favorable amino acid (lysi ne). The role of thrombin residues Arg(35), Lys(36), Glu(39) and Lys(6 0f) in the putative P-2' and P-3' binding sites was also examined. Rep lacement of Lys(60f) by glutamine improved the rate of cleavage for pe ptides with P-2' lysine or leucine. Compared with thrombin, mutants E3 9K and E39Q hydrolyzed faster substrates with an acidic residue in P-2 ' or P-3', but slightly slower those with a lysine at either position. Mutations R35Q and K36Q only improved the hydrolysis of substrates wi th an acidic P-2' residue. Overall, thrombin prefers bulky hydrophobic side chains in subsite S-2' and positively charged residues in S-3', whereas acidic residues are markedly antagonistic to both subsites.