Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase.

Arch Biochem Biophys 2006 May 27;449(1-2):1-7. Epub 2006 Mar 27.

Institute of Biomedicine, Department of Anatomy, University of Turku, Turku, Finland.

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.

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.abb.2006.03.010DOI Listing
May 2006
9 Reads

Publication Analysis

Top Keywords

ros-generating activity
16
phosphatase ros-generating
12
disulfide bond
12
redox-active iron
12
tartrate-resistant acid
8
peptide redox-active
8
loop peptide
8
increased kcat/km
8
trypsin cleavage
8
protease-sensitive loop
8
cleavage trypsin
8
ascorbate trypsin
8
acid phosphatase
8
phosphatase
6
activity
5
baculovirus-generated recombinant
4
activity baculovirus-generated
4
ascorbate phosphatase
4
iron ascorbate
4
recombinant human
4

Similar Publications