ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 246, No. 2, May 1, pp. 645-649,1986 Functional Constituents of the Active Site of Human Neutrophil Collagenase KASIM A. MOOKHTIAR, FRANK WANG, AND HAROLD E. VAN WART’ Department of Chemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32.906 Received December 9,1985 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 l,lO-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 ad- dition of hydroxylamine, indicating the presence of an essential lysine residue. 0 1986 Academic Press, Inc. Human neutrophils contain a specific collagenase’ (EC 3.4.24.7) that hydrolyzes native collagen into two characteristic fragments that are approximately three- quarters and one-quarter of its original length (1-17). Unlike most tissue collagen- ases that are synthesized by cells on stim- ulation (18), neutrophil collagenase is stored in specific granules (11). Collagen degradation is associated with both acute and chronic inflammation. Since inflamed areas contain large numbers of neutro- phils, it is likely that neutrophil collage- nase plays a central role in the connective tissue pathophysiology of this condition. i To whom correspondence should be addressed. z Abbreviations used: Unless otherwise stated, col- lagenase refers to human neutrophil collagenase. Tri- tine, tris(hydroxymethyl)methylglycine; Mes, I-mor- pholineethanesulfonic acid; Hepes, 4-(2-hydroxy- ethyl)-1-piperazine-ethanesulfonic acid; AC, acetyl; Nph, 4-nitrophenylalanine. Neutrophil collagenase is believed to be a metalloproteinase (11,19). However, little else is currently known about the active site of the enzyme. Such knowledge is nec- essary for an understanding of the mech- anism of action of the enzyme. In the pres- ent study, a series of chemical modification reactions has been carried out in order to identify functional constituents of the ac- tive site. MATERIALS AND METHODS Latent neutrophil collagenase was isolated from human blood by a modification of the procedures of Christner and co-workers (14) and Macartney and Tschesche (15). The enzyme was devoid of activity to- ward gelatin and casein and had a specific activity of 2.2 gg collagen hydrolyzed/min/mg protein, as mea- sured by the assay described below. Collagenase ac- tivity was determined by measuring the rate of hy- drolysis of “H-acetylated rat tail tendon type I collagen by a modification of the method of Ishikawa and Nimni (29). Assays were carried out at 30°C in 50 mM Tricine, 645 9003-9861/86 $3.96 Copyright 8 19%by Academic Press, Inc. All rights of repmductionin any form reserved.