Oxidative inhibition of human soluble catechol-O-methyltransferase.
A common polymorphism in the human gene for catechol-O-methyltransferase results in replacement of Val-108 by Met in the soluble form of the protein (s-COMT) and has been linked to breast cancer and neuropsychiatric disorders. The 108M and 108V variants are reported to differ in their thermal stability, with 108M COMT losing catalytic activity more rapidly. Because human s-COMT contains seven cysteine residues and includes CXXC and CXXS motifs that are associated with thiol-disulfide redox reactions, we examined the effects of reducing and oxidizing conditions on the enzyme. In the absence of a reductant 108M s-COMT lost activity more rapidly than 108V, whereas in the presence of 4 mm dithiothreitol (DTT) we found no significant differences in the stability of the two variants at 37 degrees C. DTT also restored most of the activity that was lost upon incubation at 37 degrees C in the absence of DTT. Mass spectrometry showed that cysteines 188 and 191 formed an intramolecular disulfide bond when s-COMT was incubated with oxidized glutathione, whereas cysteines 69, 95, 157, and 173 formed protein-glutathione adducts. Replacing Cys-95 by serine protected 108M s-COMT against inactivation in the absence of a reductant; C33S and Cys-188 mutations had little effect, and C69S was destabilizing. The sequences surrounding the reactive cysteine residues of human s-COMT and other proteins that form glutathione adducts at identified sites all include Pro and/or Gly and most include a hydrogen-bonding residue, suggesting that glutathiolation at conserved sites plays a physiologically important role.
Cotton
Naomi J H
NJ
Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350, USA.
Stoddard
Barry
B
Parson
William W
WW
D000595:Amino Acid Sequence; D001665:Binding Sites; D002394:Catechol O-Methyltransferase / Q000235:genetics / Q000378:metabolism; D065098:Catechol O-Methyltransferase Inhibitors; D003545:Cysteine / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D016415:Sequence Alignment
D065098:Catechol O-Methyltransferase Inhibitors; D002394:Catechol O-Methyltransferase; D005978:Glutathione; D003545:Cysteine