title: Implication of Arg213 and Arg337 on the kinetic and structural stability of firefly luciferase.
abstract: Possible roles of two different Arginine (Arg; R) 213 and 337 on kinetic and structural stability of Photinus pyralis luciferase have been investigated using thermal and chemical denaturation studies. This enzyme is highly sensitive to protease digestion and temperature, which limits its fieldability, particularly for in vivo imaging. In order to generate more stable luciferases against trypsin digestion, site-directed mutagenesis was conducted to block two representative tryptic sites on the surface of N-terminal domain, via substitution of Arg213 and Arg337 by methionine (Met; M) and glutamine (Gln; Q), respectively [A. Riahi-Madvar, S. Hosseinkhani, Protein engineering, design and selection 22 (2009) 655-663]. The improvement of mutant enzymes stability against protease hydrolysis may be attributed to the more rigidity of the enzyme structure upon mutations, as can be deducted from elevated levels of m(U-N) values and decrease of activation energy. Furthermore, mutation at position 337 which is accompanied with more alteration on the basic kinetic properties relative to mutation at position 213, revealed the high values of the ΔG(H(2)O), half-time of inactivation at 30°C and T(m) for R337Q where Arg213 is maintained in structure. Based on the results, it can be concluded that whilst Arg213 affects structural stability, Arg337 is critical for kinetic stability.
authors.affiliation: Department of Biotechnology, International Center for Science, High Technology & Environmental Sciences, Kerman, Iran. ariahi@icst.ac.ir
authors.forename: Ali
authors.initials: A
authors.lastname: Riahi-Madvar
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Rezaee
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D048888:Fireflies / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D001120:Arginine; D049409:Luciferases, Firefly