Welgus H G, Jeffrey J J, Eisen A Z, Roswit W T, Stricklin G P
Coll Relat Res. 1985 Mar;5(2):167-79. doi: 10.1016/s0174-173x(85)80038-8.
Human skin fibroblasts secrete collagen, procollagenase and a collagenase inhibitor. This study addresses the nature of the interaction between these important fibroblast products. The binding of procollagenase and of active collagenase to native collagen in solution was examined by employing Sephadex G-150 gel-filtration chromatography to separate bound versus unbound enzyme. Active enzyme bound readily to collagen; the equilibrium constant of binding, Kd, was calculated to be 5.1 to 10(-7)M. Thus, collagenase binds with nearly equal affinity to both monomeric collagen and aggregated fibrils (Kd = 9 X 10(-7)M; [Welgus et al., 1980]). Furthermore, since Kd congruent to Km congruent to 10(-6)M, the ratio k2/k1 must be extremely small, directly implicating the catalytic step represented by the rate constant k2, and not the binding of enzyme to substrate, as the rate-limiting step of collagenase action. In contrast, procollagenase demonstrated no capacity to bind to collagen. The interaction of procollagenase and of active collagenase with inhibitor was examined utilizing both conventional and high-precision liquid gel-filtration chromatography. A higher molecular weight complex could be demonstrated consisting of active collagenase and inhibitor; no such interaction occurred between procollagenase and the inhibitory protein. Analysis of Lineweaver-Burk plots showed that inhibition was accompanied by a corresponding change in Vmax; Km remained unchanged. Such results are indicative of a noncompetitive mechanism of inhibition and are consistent with the formation of an enzyme-inhibitor complex. The Ki of enzyme-inhibitor binding was determined to be less than 10(-9)M. The data indicate that procollagenase can neither interact with its specific inhibitor nor bind to collagen. Extracellular activation of the collagenase zymogen is thus a critical event, which can be followed either by binding to substrate or interaction with inhibitor.
人皮肤成纤维细胞分泌胶原蛋白、前胶原酶和一种胶原酶抑制剂。本研究探讨了这些重要的成纤维细胞产物之间相互作用的本质。通过使用葡聚糖G - 150凝胶过滤色谱法分离结合态与未结合态的酶，研究了前胶原酶和活性胶原酶与溶液中天然胶原蛋白的结合情况。活性酶很容易与胶原蛋白结合；结合的平衡常数Kd经计算为5.1×10⁻⁷M。因此，胶原酶与单体胶原蛋白和聚集的纤维原具有几乎相等的亲和力（Kd = 9×10⁻⁷M；[韦古斯等人，1980年]）。此外，由于Kd与Km相当，均约为10⁻⁶M，k2/k1的比值必定极小，这直接表明由速率常数k2代表的催化步骤，而非酶与底物的结合，是胶原酶作用的限速步骤。相比之下，前胶原酶没有显示出与胶原蛋白结合的能力。利用常规和高精度液相凝胶过滤色谱法研究了前胶原酶和活性胶原酶与抑制剂的相互作用。可以证明存在一种由活性胶原酶和抑制剂组成的更高分子量复合物；前胶原酶与抑制蛋白之间未发生这种相互作用。对Lineweaver - Burk图的分析表明，抑制伴随着Vmax的相应变化；Km保持不变。这些结果表明是一种非竞争性抑制机制，并且与酶 - 抑制剂复合物的形成一致。酶 - 抑制剂结合的Ki测定值小于10⁻⁹M。数据表明前胶原酶既不能与其特异性抑制剂相互作用，也不能与胶原蛋白结合。因此，胶原酶原的细胞外激活是一个关键事件，之后要么是与底物结合，要么是与抑制剂相互作用。
