Stephens A W, Siddiqui A, Hirs C H
Department of Biochemistry, University of Colorado Health Science Center, Denver 80262.
J Biol Chem. 1988 Nov 5;263(31):15849-52.
Human antithrombin III (AT) shares significant sequence homology and a common inhibitory mechanism with the serine protease inhibitor (serpin) superfamily. AT has a reactive site in which the P1 residue is primarily responsible for protease specificity. The P1' residue, almost invariably serine, is critical in the inactive natural variant AT-Denver, which has a leucine substitution in that position (Stephens, A.W., Thalley, B.S., and Hirs, C.H.W. (1987) J. Biol. Chem. 262, 1044-1048). In the present study site-directed mutagenesis was used to generate eight variants with altered P1' residues. All were secreted efficiently by COS cells transiently transfected with the AT cDNA in a eukaryotic shuttle vector. All variants also bound heparin as effectively as wild-type AT. Variants were grouped into three categories with respect to thrombin-AT complex formation: 1) no detectable inhibitory activity (proline, methionine); 2) low activity (cysteine, valine, leucine); and 3) near normal activity (glycine, alanine, threonine). The leucine variant, which is in the low activity group, exhibited the same physical and functional properties as AT-Denver. We conclude that the serine hydroxyl is not critical for functional activity and that there is a side chain size optimum which is modulated by hydrophobic effects.
人抗凝血酶III（AT）与丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂）超家族具有显著的序列同源性和共同的抑制机制。AT具有一个反应位点，其中P1残基主要负责蛋白酶特异性。P1'残基几乎总是丝氨酸，在无活性的天然变体AT-Denver中至关重要，该变体在该位置有一个亮氨酸取代（斯蒂芬斯，A.W.，萨利，B.S.，和赫尔斯，C.H.W.（1987年）《生物化学杂志》262，1044-1048）。在本研究中，使用定点诱变产生了八个P1'残基改变的变体。所有变体都能被用真核穿梭载体中AT cDNA瞬时转染的COS细胞有效分泌。所有变体与肝素的结合效果也与野生型AT一样好。就凝血酶-AT复合物的形成而言，变体被分为三类：1）无可检测的抑制活性（脯氨酸、甲硫氨酸）；2）低活性（半胱氨酸、缬氨酸、亮氨酸）；3）接近正常活性（甘氨酸、丙氨酸、苏氨酸）。处于低活性组的亮氨酸变体表现出与AT-Denver相同的物理和功能特性。我们得出结论，丝氨酸羟基对功能活性并不关键，并且存在一个由疏水效应调节的最佳侧链大小。
