title: High temperature biocatalysis by chemically modified cytochrome C.
abstract: Chemically modified cytochrome c with poly(ethylene glycol) (PEG) showed activity at temperatures higher than 100 degrees C and to be highly thermostable. The molecular size of PEG moieties and the coupling site affected the thermal stabilization. An optimal PEG/protein mass ratio of 2.8 was found, producing a fully thermostable biocatalyst at 80 degrees C. Site-directed mutagenesis on yeast cytochrome c showed an increased thermostabilization when lysine 79 residue, localized at the edge of the active site, was replaced by a nonreactive residue. Tertiary, secondary, and active-site structures were analyzed by fluorescence, CD, and UV/visible spectroscopies. Besides its disordered structure, the pegylated protein showed a lower unfolding rate at the active-site than the unmodified ones. A shell-like structure seems to protect the heme environment, in which PEG is coiled on the protein surface with a primary shield of rigid water molecules solvating the hydrophilic region of bound-PEG, and the PEG hydrophobic regions interacting with the hydrophobic clusters on protein surface.
authors.affiliation: Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, CP 62250 México.
authors.forename: Humberto
authors.initials: H
authors.lastname: García-Arellano
authors.forename: Brenda
authors.initials: B
authors.lastname: Valderrama
authors.forename: Gloria
authors.initials: G
authors.lastname: Saab-Rincón
authors.forename: Rafael
authors.initials: R
authors.lastname: Vazquez-Duhalt
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006736:Horses; D006358:Hot Temperature; D009206:Myocardium; D010084:Oxidation-Reduction; D011083:Polycyclic Compounds / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D017510:Protein Folding; D012441:Saccharomyces cerevisiae; D013057:Spectrum Analysis; D013816:Thermodynamics
chemical_list: D003574:Cytochrome c Group; D011083:Polycyclic Compounds; D011092:Polyethylene Glycols