Bicsak T A, Harper E
Arch Biochem Biophys. 1985 Oct;242(1):256-62. doi: 10.1016/0003-9861(85)90500-4.
The purified collagenase from tadpole (Rana catesbiana) back skin was studied with respect to its activation energy using soluble and fibrillar type I collagen, as well as a synthetic peptide substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. The activation energy appeared to be independent of the nature of the substrate, ranging between 28 and 35 kcal/mol. The peptide was cleaved at the Gly-Ile bond and proved to be a poor substrate (kcat/Km, 1.21 h-1 microM-1) when compared with native type I collagen in solution (kcat/Km, 40.6 h-1 microM-1), consistent with the enzyme's low activity versus gelatin [T. A. Bicsak and E. Harper (1984) J. Biol. Chem. 259, 13145]. The amino acid composition of the collagenase was shown to be high in glycine and glutamic acid, and the preparation was shown not to be contaminated with collagen by digestion with bacterial collagenase. The enzyme was not inhibited by iodoacetic acid or 2-hydroxy-5-nitrobenzyl bromide, suggesting the lack of essential cysteinyl and tryptophanyl residues, but was inhibited by micromolar concentrations of ZnCl2, consistent with the presence of essential histidine(s). Ethoxyformic anhydride irreversibly inhibited the collagenase suggesting the presence of essential lysyl residues.
使用可溶性和纤维状I型胶原蛋白以及合成肽底物DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg，对从牛蛙（Rana catesbiana）背部皮肤中纯化得到的胶原酶的活化能进行了研究。活化能似乎与底物的性质无关，范围在28至35千卡/摩尔之间。该肽在Gly-Ile键处被切割，与溶液中的天然I型胶原蛋白（kcat/Km，40.6 h-1 μM-1）相比，它被证明是一种较差的底物（kcat/Km，1.21 h-1 μM-1），这与该酶对明胶的低活性一致[T. A. Bicsak和E. Harper（1984年）《生物化学杂志》259，13145]。胶原酶的氨基酸组成显示甘氨酸和谷氨酸含量较高，并且通过用细菌胶原酶消化表明该制剂未被胶原蛋白污染。该酶不受碘乙酸或2-羟基-5-硝基苄基溴的抑制，表明缺乏必需的半胱氨酰和色氨酰残基，但受到微摩尔浓度的ZnCl2的抑制，这与存在必需的组氨酸一致。乙氧基甲酸酐不可逆地抑制胶原酶，表明存在必需的赖氨酰残基。
