title: Thermostability, solvent tolerance, catalytic activity and conformation of cofactor modified horseradish peroxidase.
abstract: Artificial prosthetic groups, HeminD1 and HeminD2, were designed and synthesized, which contain one benzene ring and one carboxylic group or two carboxylic groups at the terminal of each propionate side chain of hemin, respectively. HeminD1 and HeminD2 were reconstituted with apo-HRP successfully to produce the two novel HRPs, rHRP1 and rHRP2, respectively. The thermal and solvent tolerances of native and reconstituted HRPs were compared. The cofactor modification increased the thermostability both in aqueous buffer and some organic solvents, and also enhanced the tolerance of some organic solvents. To determine the conformation stability, the unfolding of native and reconstituted HRPs by heat was investigated. Tm was increased from 70.0 degrees C of nHRP to 75.4 degrees C of rHRP1 and 76.5 degrees C of rHRP2 after cofactor modification. Kinetic studies indicated that the cofactor modification increased the substrate affinity and catalytic efficiency both in aqueous buffer and some organic solvents. The catalytic efficiency for phenol oxidation was increased by approximately 55% for rHRP1 in aqueous buffer, and it was also increased by approximately 70% for rHRP1 in 10% ACN. Spectroscopic studies proved that the cofactor modification changed the microenvironment of both heme and tryptophan, increased alpha-helix content, and increased the tertiary structure around the aromatic residue in HRP. The improvements of catalytic properties are related to these changes of the conformation. The introduction of the hydrophobic domain as well as the retention of the moderate carboxylic group in active site is an efficient method to improve the thermodynamic and catalytic efficiency of HRP.
authors.affiliation: Key Laboratory of Gene Engineering of Ministry of Education and Biotechnology Research Center, State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, PR China.
authors.forename: Jiu-Ying
authors.initials: JY
authors.lastname: Feng
authors.forename: Jian-Zhong
authors.initials: JZ
authors.lastname: Liu
authors.forename: Liang-Nian
authors.initials: LN
authors.lastname: Ji
mesh_terms: D002384:Catalysis; D002942:Circular Dichroism; D004795:Enzyme Stability; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D017433:Protein Structure, Secondary; D012997:Solvents; D013696:Temperature; D013816:Thermodynamics
chemical_list: D012997:Solvents; D006735:Horseradish Peroxidase