Characterization of the P-2' and P-3' specificities of thrombin using fluorescence-quenched substrates and mapping of the subsites by mutagenesis

The importance of substrate residues P-2' and P-3' On thrombin catalysis has been investigated by comparing the hydrolysis of a series of fluorescence-quenched substrates, Each consisted of a 10-residue peptide, carrying a 2-aminobenzoyl (Abz) group at the N-terminus, and a penultimate 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' residue was found to have a major influence on the rate of cleavage: the k(cat)/K-m value for the hydrolysis of the Arg-Ser bond in Abz-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 aspartate 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 substrate with the least effective residue (aspartate) was only 33 times lower than that of the substrate with the most favorable amino acid (lysine). The role of thrombin residues Arg(35), Lys(36), Glu(39) and Lys(60f) in the putative P-2' and P-3' binding sites was also examined. Replacement of Lys(60f) by glutamine improved the rate of cleavage for peptides with P-2' lysine or leucine. Compared with thrombin, mutants E39K 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 with 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.