Kung C, Hayes E, Mann K G
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405-0068.
J Biol Chem. 1994 Oct 14;269(41):25838-48.
Prothrombinase assembly takes place on the surface of unsaturated phosphatidylcholine (PC), phosphatidylserine (PS) membranes in the presence of Ca2+, through the rapid association of membrane-bound factor Va and factor Xa. The present study uses saturated PCPS (75:25, w/w) vesicles to study prothrombinase assembly and catalytic properties in order to differentiate the influences of the membrane upon catalyst assembly, substrate delivery, and peptide bond cleavage. In contrast to studies using unsaturated phospholipid, prothrombin activation studies using saturated PCPS (75:25, w/w) (C14:0, C16:0, and C18:0) revealed up to a 20-fold decrease in prothrombinase activity. C18:0 membranes support at least 50% of the prothrombinase binding capacity (KdVa-Xa = 1 nM and nVa-Xa = 1.1) of C18:1 PCPS (75:25, w/w). Thus, the 95% loss in activity cannot be explained by gross alterations in catalyst concentration or assembly. Stopped-flow studies with saturated lipids demonstrate that factor Va, factor Xa, and prothrombin have decreased kon values. Compensatory changes in koff leave the Kd values for these protein-lipid interactions almost unchanged relative to unsaturated PCPS. The profoundly decreased activation rate on saturated phospholipid membranes as compared to unsaturated phospholipids is in part due to slowed substrate/enzyme delivery caused by the saturated lipids. However, studies using prethrombin-1 and C18:0 PCPS (75:25, w/w) also revealed a 15-fold decrease in activity for preassembled prothrombinase. Although there was a slight change in Km (+2-fold), the major cause of the decrease is an 18-fold decrease in kcat. Similar differences for Km and kcat values were obtained for prothrombin. Substrate delivery is thus only partially responsible for the diminished prothrombinase activity observed with saturated phospholipids. Since the activity of prothrombinase is decreased for both prothrombin and prethrombin-1 principally by reducing kcat, it appears that catalyst formation on saturated phospholipids somehow compromises the proteolytic activity of the enzyme complex. This implies that the phospholipid bilayer serves not merely as a surface for condensing the proteins but also as a functional element of the prothrombinase enzyme.
凝血酶原酶组装在不饱和磷脂酰胆碱（PC）、磷脂酰丝氨酸（PS）膜表面进行，在Ca2+存在下，通过膜结合的因子Va和因子Xa快速结合实现。本研究使用饱和PCPS（75:25，w/w）囊泡来研究凝血酶原酶组装和催化特性，以区分膜对催化剂组装、底物递送和肽键裂解的影响。与使用不饱和磷脂的研究不同，使用饱和PCPS（75:25，w/w）（C14:0、C16:0和C18:0）进行的凝血酶原激活研究显示，凝血酶原酶活性降低了20倍。C18:0膜支持的凝血酶原酶结合能力（KdVa-Xa = 1 nM，nVa-Xa = 1.1）至少为C18:1 PCPS（75:25，w/w）的50%。因此，活性降低95%无法用催化剂浓度或组装的总体变化来解释。对饱和脂质进行的停流研究表明，因子Va、因子Xa和凝血酶原的kon值降低。koff的补偿性变化使这些蛋白质-脂质相互作用的Kd值相对于不饱和PCPS几乎保持不变。与不饱和磷脂相比，饱和磷脂膜上激活速率大幅降低部分是由于饱和脂质导致底物/酶递送减慢。然而，使用凝血酶原-1和C18:0 PCPS（75:25，w/w）进行的研究也显示，预组装的凝血酶原酶活性降低了15倍。尽管Km有轻微变化（增加2倍），但活性降低的主要原因是kcat降低了18倍。凝血酶原的Km和kcat值也有类似差异。因此，底物递送只是饱和磷脂导致凝血酶原酶活性降低的部分原因。由于凝血酶原酶对凝血酶原和凝血酶原-1的活性主要通过降低kcat而降低，似乎饱和磷脂上的催化剂形成以某种方式损害了酶复合物的蛋白水解活性。这意味着磷脂双层不仅作为浓缩蛋白质的表面，还作为凝血酶原酶的功能元件。
