Calugaru S V, Swanson R, Olson S T
Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
J Biol Chem. 2001 Aug 31;276(35):32446-55. doi: 10.1074/jbc.M104731200. Epub 2001 Jun 12.
Serpin family protein proteinase inhibitors trap proteinases at the acyl-intermediate stage of cleavage of the serpin as a proteinase substrate by undergoing a dramatic conformational change, which is thought to distort the proteinase active site and slow deacylation. To investigate the extent to which proteinase catalytic function is defective in the serpin-proteinase complex, we compared the pH dependence of dissociation of several serpin-proteinase acyl-complexes with that of normal guanidinobenzoyl-proteinase acyl-intermediate complexes. Whereas the apparent rate constant for dissociation of guanidinobenzoyl-proteinase complexes (k(diss, app)) showed a pH dependence characteristic of His-57 catalysis of complex deacylation, the pH dependence of k(diss, app) for the serpin-proteinase complexes showed no evidence for His-57 involvement in complex deacylation and was instead characteristic of a hydroxide-mediated deacylation similar to that observed for the hydrolysis of tosylarginine methyl ester. Hydroxylamine enhanced the rate of serpin-proteinase complex dissociation but with a rate constant for nucleophilic attack on the acyl bond several orders of magnitude slower than that of hydroxide, implying limited accessibility of the acyl bond in the complex. The addition of 10-100 mm Ca(2+) ions stimulated up to 80-fold the dissociation rate constant of several serpin-trypsin complexes in a saturable manner at neutral pH and altered the pH dependence to a pattern characteristic of His-57-catalyzed complex deacylation. These results support a mechanism of kinetic stabilization of serpin-proteinase complexes wherein the complex is trapped as an acyl-intermediate by a serpin conformational change-induced inactivation of the proteinase catalytic function, but suggest that the inactive proteinase conformation in the complex is in equilibrium with an active proteinase conformation that can be stabilized by the preferential binding of an allosteric ligand such as Ca(2+).
丝氨酸蛋白酶抑制剂家族蛋白作为蛋白酶底物时，通过发生剧烈的构象变化，在丝氨酸蛋白酶抑制剂裂解的酰基中间体阶段捕获蛋白酶，这种构象变化被认为会扭曲蛋白酶活性位点并减缓脱酰基作用。为了研究蛋白酶催化功能在丝氨酸蛋白酶抑制剂 - 蛋白酶复合物中缺陷的程度，我们比较了几种丝氨酸蛋白酶抑制剂 - 蛋白酶酰基复合物解离的pH依赖性与正常胍基苯甲酰 - 蛋白酶酰基中间体复合物的pH依赖性。胍基苯甲酰 - 蛋白酶复合物的表观解离速率常数（k(diss, app)）呈现出His - 57催化复合物脱酰基作用的pH依赖性特征，而丝氨酸蛋白酶抑制剂 - 蛋白酶复合物的k(diss, app)的pH依赖性没有显示出His - 57参与复合物脱酰基作用的证据，而是类似于对甲苯磺酰精氨酸甲酯水解所观察到的氢氧化物介导的脱酰基作用特征。羟胺提高了丝氨酸蛋白酶抑制剂 - 蛋白酶复合物的解离速率，但对酰基键亲核攻击的速率常数比对氢氧化物慢几个数量级，这意味着复合物中酰基键的可及性有限。在中性pH下，添加10 - 100 mM Ca(2+)离子以饱和方式刺激了几种丝氨酸蛋白酶抑制剂 - 胰蛋白酶复合物的解离速率常数高达80倍，并将pH依赖性改变为His - 57催化复合物脱酰基作用的特征模式。这些结果支持了丝氨酸蛋白酶抑制剂 - 蛋白酶复合物动力学稳定的机制，其中复合物通过丝氨酸蛋白酶抑制剂构象变化诱导的蛋白酶催化功能失活而被捕获为酰基中间体，但表明复合物中无活性的蛋白酶构象与活性蛋白酶构象处于平衡状态，活性蛋白酶构象可通过变构配体如Ca(2+)的优先结合而稳定。
