Mookhtiar K A, Wang F, Van Wart H E
Arch Biochem Biophys. 1986 May 1;246(2):645-9. doi: 10.1016/0003-9861(86)90320-6.
A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.
已经进行了一系列化学修饰反应，以鉴定人中性粒细胞胶原酶活性位点的功能成分。该酶被过渡金属螯合剂1,10 - 菲咯啉可逆抑制，并且锌可完全逆转抑制作用。通过凝胶过滤去除弱结合的金属离子会使胶原酶失活，而立即重新添加钙后活性可完全恢复。该酶不受修饰丝氨酸、半胱氨酸和精氨酸残基的试剂影响。然而，与羧基试剂环己基吗啉代碳二亚胺和伍德沃德试剂K反应会大幅降低该酶的活性。乙酰咪唑会使该酶失活，但添加羟胺后活性可完全恢复。该酶也会被四硝基甲烷灭活，表明它含有一个必需的酪氨酸残基。用焦碳酸二乙酯、双乙烯酮、乙酸酐或三硝基苯磺酸对胶原酶进行酰化会使该酶失活，添加羟胺后活性无法恢复，表明存在一个必需的赖氨酸残基。
