Functional constituents of the active site of human neutrophil collagenase.
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.
D000596:Amino Acids / Q000097:blood; D001665:Binding Sites; D004793:Enzyme Reactivators; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008239:Lysine / Q000097:blood; D008670:Metals / Q000097:blood; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000097:blood*; D009504:Neutrophils / Q000201:enzymology*; D015125:Oxyquinoline / Q000031:analogs & derivatives / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D014443:Tyrosine / Q000097:blood; D015032:Zinc / Q000494:pharmacology
D000596:Amino Acids; D004793:Enzyme Reactivators; D006898:Hydroxylamines; D007093:Imidazoles; D008670:Metals; D010618:Phenanthrolines; D019811:Hydroxylamine; D014443:Tyrosine; D015125:Oxyquinoline; C013053:8-hydroxyquinoline-5-sulfonic acid; D003012:Microbial Collagenase; D015032:Zinc; D008239:Lysine; C025205:1,10-phenanthroline; C012160:N-acetylimidazole