Hasty K A, Jeffrey J J, Hibbs M S, Welgus H G
J Biol Chem. 1987 Jul 25;262(21):10048-52.
The substrate specificity of human neutrophil collagenase was examined using both monomeric and fibrillar collagens. The neutrophil enzyme cleaved types I, II, and III collagens, but failed to attack types IV or V. Against monomeric collagen substrates at 25 degrees C, the neutrophil enzyme displayed values for the Michaelis constant (Km) of 0.6-1.8 X 10(-6) M, essentially indistinguishable from the substrate affinities that characterize human fibroblast collagenase. Catalytic rates, however, varied considerably; type I collagen was cleaved with a specificity (kappa cat/Km) some 20-fold greater than type III. Type II collagen was degraded with intermediate selectivity, approximately equal to 25% of the type I rate, but 450% that of type III. This specificity contrasted markedly with that of human fibroblast collagenase, which cleaved human type III collagen 15-fold faster than type I and greater than 500-fold more rapidly than type II. Interestingly, the 20-fold selectivity for type I over type III exhibited by neutrophil collagenase against monomeric collagens was largely abolished following the reconstitution of these substrates into insoluble fibrils, falling to a value of just 1.5-fold. The distinctive and opposite preference by the human fibroblast enzyme for monomeric type III collagen over type I (15-fold) was similarly reduced to less than 2-fold upon substrate aggregation. The transition from native soluble collagen monomers into insoluble fibrils appeared to be handled by both the human neutrophil and fibroblast collagenases with similar facility on type I substrates. By comparison, however, the neutrophil enzyme degraded type III collagen fibrils faster than would have been predicted from solution rates, while the fibroblast enzyme cleaved such fibrils much slower than expected from solution values. In exploring this phenomenon further, solvent deuterium isotope effects were measured. The deuterium studies suggest that neutrophil collagenase, acting on type III fibrils (kappa H2O/kappa D2O = 5.0), is less sensitive to factors which govern the availability of water at the relatively hydrophobic site of peptide bond hydrolysis in the collagen molecule than is fibroblast collagenase (kappa H2O/kappa D2O = 15.0).
利用单体胶原和原纤维胶原对人中性粒细胞胶原酶的底物特异性进行了检测。中性粒细胞酶可切割I型、II型和III型胶原，但不能作用于IV型或V型胶原。在25℃下，针对单体胶原底物，中性粒细胞酶的米氏常数（Km）值为0.6 - 1.8×10⁻⁶ M，与表征人成纤维细胞胶原酶的底物亲和力基本无法区分。然而，催化速率差异很大；I型胶原的切割特异性（催化常数/米氏常数）比III型胶原约高20倍。II型胶原以中等选择性被降解，约为I型胶原降解速率的25%，但却是III型胶原降解速率的450%。这种特异性与人成纤维细胞胶原酶的特异性形成显著对比，后者切割人III型胶原的速度比I型胶原快15倍，比II型胶原快500倍以上。有趣的是，中性粒细胞胶原酶对单体胶原中I型胶原相对于III型胶原的20倍选择性，在这些底物重构为不溶性原纤维后基本消失，降至仅1.5倍。人成纤维细胞酶对单体III型胶原相对于I型胶原的独特且相反的偏好（15倍）在底物聚集后同样降至不到2倍。从天然可溶性胶原单体向不溶性原纤维的转变，人中性粒细胞胶原酶和成纤维细胞胶原酶在I型底物上似乎都能以类似的能力处理。然而，相比之下，中性粒细胞酶降解III型胶原原纤维的速度比根据溶液速率预测的要快，而成纤维细胞酶切割此类原纤维的速度比根据溶液值预期的要慢得多。为了进一步探究这一现象，测量了溶剂氘同位素效应。氘研究表明，作用于III型原纤维的中性粒细胞胶原酶（H₂O催化常数/D₂O催化常数 = 5.0），与成纤维细胞胶原酶（H₂O催化常数/D₂O催化常数 = 15.0）相比，对控制胶原分子中肽键水解相对疏水部位水可用性的因素不太敏感。
