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 tetranitromethane. 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(e1)) 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.
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Yuko
Y
Muta
Hiroshi
H
Oneda
Kuniyo
K
Inouye
D019297:2,4-Dinitrophenol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000586:Amination; D024342:Amino Acids, Acidic / Q000737:chemistry / Q000378:metabolism; D001391:Azo Compounds / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism / Q000379:methods; D003094:Collagen / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010958:Plasminogen; D011994:Recombinant Proteins; D000637:Transaminases / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism*
D024342:Amino Acids, Acidic; D001391:Azo Compounds; D009842:Oligopeptides; C118420:PLGLB1 protein, human; D011994:Recombinant Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine; C028470:Azocoll; D010958:Plasminogen; D003094:Collagen; D000637:Transaminases; D020783:Matrix Metalloproteinase 7; D019297:2,4-Dinitrophenol