Chen V C, Chao L, Chao J
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2000 Dec 8;275(49):38457-66. doi: 10.1074/jbc.M005605200.
Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.
激肽释放酶抑制蛋白是一种具有独特P1苯丙氨酸的丝氨酸蛋白酶抑制剂，对组织激肽释放酶具有优异的抑制特异性。在本研究中，我们通过定点诱变和分子建模研究了人激肽释放酶抑制蛋白的P3 - P2 - P1残基（第386 - 388位残基）在决定对人组织激肽释放酶的抑制特异性方面的作用。构建并纯化了在每个P1、P2或P3残基处有19种不同氨基酸取代的人激肽释放酶抑制蛋白突变体，以比较它们与激肽释放酶的结合活性。复合物形成分析表明，在P1变体中，P1精氨酸、P1苯丙氨酸（野生型）、P1赖氨酸、P1酪氨酸、P1甲硫氨酸和P1亮氨酸与组织激肽释放酶表现出显著的结合活性。动力学分析表明，P1突变体对组织激肽释放酶的抑制活性顺序为P1精氨酸 > P1苯丙氨酸 > P1赖氨酸 >= P1酪氨酸 > P1亮氨酸 >= P1甲硫氨酸。由于P1精氨酸还抑制其他几种丝氨酸蛋白酶，所以P1苯丙氨酸对人组织激肽释放酶表现出比P1精氨酸更好的选择性。肝素可区分激肽释放酶抑制蛋白对激肽释放酶和胰凝乳蛋白酶的抑制特异性。对于P2和P3变体，在P2处具有疏水且体积较大氨基酸以及在P3处具有碱性氨基酸的突变体与组织激肽释放酶表现出更好的结合活性。这些突变体对组织激肽释放酶的抑制活性顺序为P2苯丙氨酸（野生型）> P2亮氨酸 > P2色氨酸 > P2甲硫氨酸以及P3精氨酸 > P3赖氨酸（野生型）。与组织激肽释放酶的活性裂隙结合的激肽释放酶抑制蛋白反应中心环的分子建模表明，P2残基需要一个长且体积大的疏水侧链才能到达并填充由组织激肽释放酶的Tyr(99)和Trp(219)产生的疏水S2裂隙。P3处的碱性氨基酸可通过与组织激肽释放酶的Asp(98J)形成静电相互作用以及与Gln(174)形成氢键来稳定复合物的形成。我们的结果表明，组织激肽释放酶是激肽释放酶抑制蛋白的特异性靶标蛋白酶。
