Welgus H G, Fliszar C J, Seltzer J L, Schmid T M, Jeffrey J J
Department of Medicine, Jewish Hospital, Washington University Medical Center, St. Louis, Missouri 63110.
J Biol Chem. 1990 Aug 15;265(23):13521-7.
We have studied the degradation of type X collagen by human skin fibroblast and rat uterus interstitial collagenases and human 72-kDa type IV collagenase. The interstitial collagenases attacked the native type X helix at two loci, cleaving residues Gly92-Leu93 and Gly420-Ile421, both scissions involving Gly-X bonds of Gly-X-Y-Z-A sequences. However, the human and rat interstitial enzymes displayed an opposite and substantial selectivity for each of these potential sites, with the uterine enzyme catalyzing the Gly420-Ile421 cleavage almost 20-fold faster than the Gly92-Leu93 locus. Values for enzyme-substrate affinity were approximately 1 microM indistinguishable from the corresponding Km values against type I collagen. Interestingly, in attacking type X collagen, both enzymes manifested kinetic properties intermediate between those characterizing the degradation of native and denatured collagen substrates. Thus, energy dependence of reaction velocity revealed a value of EA of 45 kcal, typical of native interstitial collagen substrates. However, the substitution of D2O for H2O in solvent buffer failed to slow type X collagenolysis significantly (kH/kD = 1.1), in contrast to the 50-70% slowing (kH/kD = 2-3) observed with native interstitial collagens. Since this lack of deuterium isotope effect is characteristic of interstitial collagenase cleavage of denatured collagens, we investigated the capacity of another metalloproteinase with substantial gelatinolytic activity, 72-kDa type IV collagenase, to degrade type X collagen. The 72-kDa type IV collagenase cleaved type X collagen at both 25 and 37 degrees C, and at loci in close proximity to those attacked by the interstitial enzymes. No further cleavages were observed at either temperature with type IV collagenase, and although values for kcat were not determined (due to associated tissue inhibitor of metalloproteinases-2), catalytic rates appeared to be substantial in comparison to the interstitial enzymes. In contrast, type X collagen was completely resistant to proteolysis by stromelysin. Type X collagen thus appears to be highly unusual in its susceptibility to degradation by both interstitial collagenase and another member of the metalloproteinase gene family.
我们研究了人皮肤成纤维细胞、大鼠子宫间质胶原酶和人72 kDa IV型胶原酶对X型胶原的降解作用。间质胶原酶在两个位点攻击天然X型螺旋，切割甘氨酸92 - 亮氨酸93和甘氨酸420 - 异亮氨酸421残基，这两个切割位点均涉及甘氨酸 - X - Y - Z - A序列中的甘氨酸 - X键。然而，人和大鼠的间质酶对这些潜在位点中的每一个都表现出相反且显著的选择性，子宫酶催化甘氨酸420 - 异亮氨酸421切割的速度比甘氨酸92 - 亮氨酸93位点快近20倍。酶 - 底物亲和力值约为1 microM，与针对I型胶原的相应Km值无明显差异。有趣的是，在攻击X型胶原时，两种酶都表现出介于天然和变性胶原底物降解特征之间的动力学特性。因此，反应速度的能量依赖性显示EA值为45千卡，这是天然间质胶原底物的典型值。然而，与天然间质胶原观察到的50 - 70%的减慢（kH/kD = 2 - 3）相反，在溶剂缓冲液中用重水（D2O）替代水未能显著减慢X型胶原的降解（kH/kD = 1.1）。由于这种缺乏氘同位素效应是变性胶原间质胶原酶切割的特征，我们研究了另一种具有大量明胶溶解活性的金属蛋白酶，即72 kDa IV型胶原酶，降解X型胶原的能力。72 kDa IV型胶原酶在25℃和37℃时均能切割X型胶原，且切割位点与间质酶攻击的位点相近。在这两个温度下，IV型胶原酶均未观察到进一步的切割，尽管未测定kcat值（由于存在金属蛋白酶组织抑制剂 - 2），但与间质酶相比，催化速率似乎相当可观。相比之下，X型胶原对基质金属蛋白酶完全具有抗蛋白水解作用。因此，X型胶原在对间质胶原酶和金属蛋白酶基因家族的另一个成员的降解敏感性方面似乎非常不同寻常。
