title: Mutation of Val90 to His in the pseudoperoxidase from Leishmania major enhances peroxidase activity.
abstract: Pseudoperoxidase from Leishmania major (LmPP) catalyzes the breakdown of peroxynitrite though it can hardly react with H(2)O(2). Our modeling structure predicts that a conserved His to Val switch near the distal heme pocket of LmPP may determine the profile of its H(2)O(2) activity. To test this hypothesis, we have generated complementary mutations in the LmPP (V90H) and studied the formation of Compounds I and II. The rate of transition from high spin ferric state of V90H to Compound I by H(2)O(2) is increased by approximately three orders relative to wild-type LmPP, which is consistent with electron donor oxidation data where the V90H mutant enzyme is ~30 fold more active than wild type. Thus, our data indicate that a lower rate for heterolytic cleavage of the OO bond of H(2)O(2) in wild type LmPP is caused by the His/Val switch in heme distal site. In the catalysis of peroxynitrite scavenging, V90H LmPP has lower catalytic activity compared to the wild type enzyme. In contrast to peroxynitrite scavenging, the second order rate constant of peroxynitrite binding step in mutant enzyme does not change significantly compared to the wild-type. Spectral data suggest that the distal Val90 residue in LmPP prevents the ferryl species formation in the presence of peroxynitrite. The lower peroxynitrite scavenging activity of the mutant reflects increased peroxidase activity rather than isomerase activity.
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700 032, India.
authors.forename: Rina
authors.initials: R
authors.lastname: Saha
authors.forename: Moumita
authors.initials: M
authors.lastname: Bose
authors.forename: Subrata
authors.initials: S
authors.lastname: Adak
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014633:Valine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D015800:Protozoan Proteins; D030421:Peroxynitrous Acid; D006639:Histidine; D006861:Hydrogen Peroxide; D009195:Peroxidase; C580840:pseudoperoxidase, Leishmania major; D014633:Valine