Critical role of Glu175 on stability and folding of bacterial luciferase: stopped-flow fluorescence study.
Bacterial luciferase is a heterodimeric enzyme, which catalyzes the light emission reaction, utilizing reduced FMN (FMNH2), a long chain aliphatic aldehyde and O(2), to produce green-blue light. This enzyme can be readily classed as slow or fast decay based on their rate of luminescence decay in a single turnover. Mutation of Glu175 in alpha subunit to Gly converted slow decay Xenorhabdus Luminescence luciferase to fast decay one. The following studies revealed that changing the luciferase flexibility and lake of Glu-flavin interactions are responsible for the unusual kinetic properties of mutant enzyme. Optical and thermodynamics studies have caused a decrease in free energy and anisotropy of mutant enzyme. Moreover, the role of Glu175 in transition state of folding pathway by use of stopped-flow fluorescence technique has been studied which suggesting that Glu175 is not involved in transition state of folding and appears as surface residue of the nucleus or as a member of one of a few alternative folding nuclei. These results suggest that mutation of Glu175 to Gly extended the structure of Xenorhabdus Luminescence luciferase, locally.
Shirazy
Najmeh Hadizadeh
NH
Department of Biophysics and Biochemistry, Tarbiat Modares University, P.O. Box: 14115-175, Tehran, Iran.
Ranjbar
Bijan
B
Hosseinkhani
Saman
S
Khalifeh
Khosrow
K
Madvar
Ali Riahi
AR
Naderi-Manesh
Hossein
H
D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D018698:Glutamic Acid / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D049453:Luciferases, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D020637:Photorhabdus / Q000187:drug effects / Q000201:enzymology*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics
D050505:Mutant Proteins; D018698:Glutamic Acid; D049453:Luciferases, Bacterial; D019791:Guanidine