Al-Ayyoubi Maher, Schwartz Bradford S, Gettins Peter G W
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
J Biol Chem. 2007 Jul 6;282(27):19502-9. doi: 10.1074/jbc.M702445200. Epub 2007 May 16.
Maspin is a member of the serpin family with a reactive center loop that is incompatible with proteinase inhibition by the serpin conformational change mechanism. Despite this there are reports that maspin might regulate uPA-dependent processes in vivo. Using exogenous and endogenous fluorescence, we demonstrate here that maspin can bind uPA and tPA in both single-chain and double-chain forms, with K(d) values between 300 and 600 nM. Binding is at an exosite on maspin close to, but outside of, the reactive center loop and is therefore insensitive to mutation of Arg(340) within the reactive center loop. The binding site on tPA does not involve the proteinase active site, with the result that maspin can bind to S195A tPA that is already complexed to plasminogen activator inhibitor-1. The ability of maspin to bind these proteinases without involvement of the reactive center loop leaves the latter free to engage in additional, as yet unidentified, maspin-protein interactions that may serve to regulate the properties of the exosite-bound proteinase. This may help to reconcile apparently conflicting studies that demonstrate the importance of the reactive center loop in certain maspin functions, despite the inability of maspin to directly inhibit tPA or uPA catalytic activity in in vitro assays through engagement between its reactive center loop and the active site of the proteinase.
Maspin是丝氨酸蛋白酶抑制剂（serpin）家族的成员，其反应中心环与通过serpin构象变化机制抑制蛋白酶不相容。尽管如此，有报道称maspin可能在体内调节依赖尿激酶型纤溶酶原激活物（uPA）的过程。利用外源性和内源性荧光，我们在此证明maspin可以结合单链和双链形式的uPA和组织型纤溶酶原激活物（tPA），解离常数（K(d)）值在300至600 nM之间。结合发生在maspin上靠近反应中心环但在其外部的一个别构部位，因此对反应中心环内的精氨酸（Arg(340)）突变不敏感。tPA上的结合位点不涉及蛋白酶活性位点，结果maspin可以结合已经与纤溶酶原激活物抑制剂-1形成复合物的S195A tPA。maspin在不涉及反应中心环的情况下结合这些蛋白酶的能力，使得后者能够自由地参与其他尚未确定的maspin-蛋白质相互作用，这些相互作用可能有助于调节别构部位结合的蛋白酶的特性。这可能有助于调和一些明显相互矛盾的研究结果，这些研究表明反应中心环在某些maspin功能中很重要，尽管在体外实验中maspin无法通过其反应中心环与蛋白酶活性位点的结合直接抑制tPA或uPA的催化活性。
