Le Bonniec B F, Myles T, Johnson T, Knight C G, Tapparelli C, Stone S R
Department of Haematology, University of Cambridge, England.
Biochemistry. 1996 Jun 4;35(22):7114-22. doi: 10.1021/bi952701s.
The importance of substrate residues P2' and P3' 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 P2' residue was found to have a major influence on the rate of cleavage: the Kcat/Km 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 P2' position. Comparatively, the P3' side chain was less important: the kcat/Km 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 Arg35, Lys36, Glu39 and Lys60f in the putative P2' and P3' binding sites was also examined. Replacement of Lys60f by glutamine improved the rate of cleavage for peptides with P2' lysine or leucine. Compared with thrombin, mutants E39K and E39Q hydrolyzed faster substrates with an acidic residue in P2' or P3', but slightly slower those with a lysine at either position. Mutations R35Q and K36Q only improved the hydrolysis of substrates with an acidic P2' residue. Overall, thrombin prefers bulky hydrophobic side chains in subsite S2' and positively charged residues in S3', whereas acidic residues are markedly antagonistic to both subsites.
通过比较一系列荧光猝灭底物的水解情况，研究了底物残基P2'和P3'对凝血酶催化作用的重要性。每个底物都由一个10个残基的肽组成，该肽在N端带有一个2-氨基苯甲酰（Abz）基团，以及一个倒数第二个2,4-二硝基苯基（Dnp）衍生的赖氨酸。这种肽的切割会解除分子内猝灭的荧光，从而可以确定动力学参数。发现P2'残基的性质对切割速率有主要影响：Abz-Val-Gly-Pro-Arg-Ser-Phe-Leu-Leu-Lys(Dnp)-Asp-OH中Arg-Ser键水解的Kcat/Km值比在P2'位置用天冬氨酸代替苯丙氨酸的相同底物水解的Kcat/Km值高近3个数量级。相比之下，P3'侧链的重要性较低：具有最无效残基（天冬氨酸）的底物的kcat/Km值仅比具有最有利氨基酸（赖氨酸）的底物低33倍。还研究了凝血酶残基Arg35、Lys36、Glu39和Lys60f在假定的P2'和P3'结合位点中的作用。用谷氨酰胺取代Lys60f提高了具有P2'赖氨酸或亮氨酸的肽的切割速率。与凝血酶相比，突变体E39K和E39Q水解P2'或P3'带有酸性残基的底物更快，但水解这两个位置带有赖氨酸的底物略慢。突变R35Q和K36Q仅改善了P2'带有酸性残基的底物的水解。总体而言，凝血酶更喜欢亚位点S'2中的大体积疏水侧链和S'3中的带正电荷残基，而酸性残基对这两个亚位点均具有明显的拮抗作用。
