title: Kinetic evidence for surface residues influencing the active site of Coprinus cinereus peroxidase: analysis of the pH dependence of G154E, P90H and P90H-G154E substrate entrance mutants.
abstract: Three mutants of Coprinus cinereus peroxidase (CIP) were made to mimic the substrate entrance histidine 82-glutamic acid 146 pair of the substrate channel in lignin peroxidase (LIP). Compound I formation of LIP has a low pH optimum around pH 3, while optimal formation of CIP compound I is obtained at pH 6-11. The mutants were glycine 154-->glutamic acid (G154E), proline 90-->histidine (P90H) and the double mutant P90H-G154E. All three showed kinetics of compound I formation similar to that of wt CIP between pH 3 and 9. However, the stability of compound I was strongly affected by these mutations. In wt CIP compound I is stable for approximately 30 min, while compound I of the mutants were stable for 5 s or less. The P90H and P90H-G154E mutants showed pK(a) values for the alkaline transition at least one pH unit lower than for wt CIP and the G154E mutant. We suggest that the changed electrostatic field results in destabilisation of the oxidised heme in compound I and II and that the P90H residue increases the electrostatic potential in the distal cavity thereby decreasing the pK(a) for the alkaline transition.
authors.affiliation: Department of Protein Chemistry, University of Copenhagen, Ø. Farimagsgade 2A, DK-1353 K, Copenhagen, Denmark.
authors.forename: P
authors.initials: P
authors.lastname: Di Cerbo
authors.forename: K G
authors.initials: KG
authors.lastname: Welinder
authors.forename: C B
authors.initials: CB
authors.lastname: Schiødt
mesh_terms: D001665:Binding Sites; D003302:Coprinus / Q000201:enzymology*; D004795:Enzyme Stability; D006418:Heme / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D006418:Heme; D006861:Hydrogen Peroxide; D010544:Peroxidases; C042858:lignin peroxidase