Anomalous pH-dependence of the activity of human matrilysin (matrix metalloproteinase-7) as revealed by nitration and amination of its tyrosine residues

Matrilysin activity exhibits a broad bell-shaped pH-dependence profile, with pK(a) values of 4.0 and 9.8. A maximum of five out of eight tyrosine residues in matrilysin were nitrated with tetra-nitromethane. On nitration of between one and five tyrosines, pK(a) at the alkaline side (pK(e2)) was shifted from 9.8 to 10.3-10.6, while that at the acidic side (pK(et)) was not altered. The pK(e2) that was shifted by nitration to 10.3-10.6 was restored to 9.4-9.7 by subsequent amination, suggesting that the shift in pK(e2) is induced. by a negative charge introduced on the most reactive tyrosine, Tyr-150. The Michaelis constant (K-m) observed at pH 10-was decreased by nitration as a result of the increase in pK(e2), suggesting that the residue with pK(e2) may play a role in the recognition of substrate. When four or five tyrosines were nitrated, the activity at pH < 7 decreased significantly, while that at pH 7-10 was unchanged, and thus the pH-dependence was not bell-shaped, but anomalous, with a third pK(a) (pK(e3)) of 6.2-6.4 in addition to pK(e1) and pK(e2). This suggests the possibility that a newly introduced nitrotyrosine residue has a strong influence on the activity as an ionizable group.