title: Polymorphisms in human soluble epoxide hydrolase: effects on enzyme activity, enzyme stability, and quaternary structure.
abstract: Human soluble epoxide hydrolase (hsEH) has been shown to play a role in regulating blood pressure and inflammation. HsEH consists of an N-terminal phosphatase and a C-terminal epoxide hydrolase domain. In the present study, we examined the effects of polymorphisms in the hsEH gene on phosphatase activity, enzyme stability, and protein quaternary structure. The results showed that mutants Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the Arg103Cys/Arg287Gln (double mutant) have significantly lower phosphatase activity compared to the most frequent allele (MFA) of hsEH. In addition, the Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the double mutant have significantly lower kcat/Km values. The stabilities at 37 degrees C of purified Arg287Gln and Arg103Cys/Arg287Gln mutants were also significantly reduced compared to the MFA. HPLC size-exclusion studies showed that the MFA exists predominantly as a dimer. However, the Arg287Gln and Arg103Cys/Arg287Gln mutants show increased concentration of the monomer. We conclude that the Arg287Gln polymorphism disrupts putative intra- and inter-monomeric salt-bridges responsible for dimerization.
authors.affiliation: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, USA.
authors.forename: Punit K
authors.initials: PK
authors.lastname: Srivastava
authors.forename: Vikas K
authors.initials: VK
authors.lastname: Sharma
authors.forename: Davendra S
authors.initials: DS
authors.lastname: Kalonia
authors.forename: David F
authors.initials: DF
authors.lastname: Grant
mesh_terms: D019943:Amino Acid Substitution; D004789:Enzyme Activation; D004795:Enzyme Stability; D004851:Epoxide Hydrolases / Q000737:chemistry*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D004851:Epoxide Hydrolases