Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase.
Osteoclasts and macrophages express high amounts of tartrate-resistant acid phosphatase (TRACP), an enzyme with unknown biological function. TRACP contains a disulfide bond, a protease-sensitive loop peptide, and a redox-active iron that can catalyze formation of reactive oxygen species (ROS). We studied the effects of proteolytic cleavage by trypsin, reduction of the disulfide bond by beta-mercaptoethanol, and reduction of the redox-active iron by ascorbate on the phosphatase and ROS-generating activity of baculovirus-generated recombinant human TRACP. Ascorbate alone and trypsin in combination with beta-mercaptoethanol increased k(cat)/K(m) of the phosphatase activity seven- to ninefold. The pH-optimum was changed from 5.4-5.6 to 6.2-6.4 by ascorbate and trypsin cleavage. Trypsin cleavage increased k(cat)/K(m) of the ROS-generating activity 2.5-fold without affecting the pH-optimum (7.0). These results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.
Fagerlund
Katja M
KM
Institute of Biomedicine, Department of Anatomy, University of Turku, Turku, Finland. katja.fagerlund@utu.fi
Ylipahkala
Hannele
H
Tiitinen
Sari L
SL
Janckila
Anthony J
AJ
Hamilton
Susan
S
Mäentausta
Olli
O
Väänänen
H Kalervo
HK
Halleen
Jussi M
JM
D000135:Acid Phosphatase / Q000737:chemistry*; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry*; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase
D007527:Isoenzymes; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases; D000071681:Tartrate-Resistant Acid Phosphatase; D010447:Peptide Hydrolases