title: Characterization of a large subunit catalase truncated by proteolytic cleavage.
abstract: The large subunit catalase HPII from Escherichia coli can be truncated by proteolysis to a structure similar to small subunit catalases. Mass spectrometry analysis indicates that there is some heterogeneity in the precise cleavage sites, but approximately 74 N-terminal residues, 189 C-terminal residues, and a 9-11-residue internal fragment, including residues 298-308, are removed. Crystal structure refinement at 2.8 A reveals that the tertiary and quaternary structure of the native enzyme is retained with only very subtle changes despite the loss of 36% of the sequence. The truncated variant exhibits a 1.8 times faster turnover rate and enhanced sensitivity to high concentrations of H(2)O(2), consistent with easier access of the substrate to the active site. In addition, the truncated variant is more sensitive to inhibition, particularly by reagents such as aminotriazole and azide which are larger than substrate H(2)O(2). The main channel leading to the heme cavity is largely unaffected by the truncation, but the lateral channel is shortened and its entrance widened by removal of the C-terminal domain, providing an explanation for easier access to the active site. Opening of the entrance to the lateral channel also opens the putative NADPH binding site, but NADPH binding could not be demonstrated. Despite the lack of bound NADPH, the compound I species of both native and truncated HPII are reduced back to the resting state with compound II being evident in the absorbance spectrum only of the heme b-containing H392A variant.
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
authors.forename: Prashen
authors.initials: P
authors.lastname: Chelikani
authors.forename: Xavier
authors.initials: X
authors.lastname: Carpena
authors.forename: Rosa
authors.initials: R
authors.lastname: Perez-Luque
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Donald
authors.forename: Harry W
authors.initials: HW
authors.lastname: Duckworth
authors.forename: Jacek
authors.initials: J
authors.lastname: Switala
authors.forename: Ignacio
authors.initials: I
authors.lastname: Fita
authors.forename: Peter C
authors.initials: PC
authors.lastname: Loewen
mesh_terms: D001665:Binding Sites; D002374:Catalase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D006418:Heme / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D006418:Heme; D006861:Hydrogen Peroxide; C102664:hydroperoxidase II; D002374:Catalase; D010447:Peptide Hydrolases