Histidine-114 at Subsites E and F Can Explain the Characteristic Enzymatic Activity of Guinea Hen Egg-white Lysozyme
| Autor: | Gen Toshima, Tomohiro Araki, Takao Torikata, Shunsuke Kawamura |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Glycosylation
Pentamer Stereochemistry Molecular Sequence Data Mutant chemical and pharmacologic phenomena Arginine Applied Microbiology and Biotechnology Biochemistry Catalysis Acetylglucosamine Analytical Chemistry chemistry.chemical_compound Egg White Animals Histidine Amino Acid Sequence Binding site Site-directed mutagenesis Molecular Biology Binding Sites Sequence Homology Amino Acid Organic Chemistry Hydrogen Bonding General Medicine Kinetics Amino Acid Substitution chemistry Mutagenesis Site-Directed Thermodynamics Muramidase Lysozyme Chickens Sequence Alignment Protein Binding Biotechnology Egg white |
| Zdroj: | Bioscience, Biotechnology, and Biochemistry. 67:540-546 |
| ISSN: | 1347-6947 0916-8451 |
| DOI: | 10.1271/bbb.67.540 |
| Popis: | The courses of the reaction catalyzed by guinea hen egg-white lysozyme (GHL), in which Asn113 and Arg114 at subsites E and F in hen egg-white lysozyme (HEL) are replaced by Lys and His, respectively, was studied with the substrate N-acetylglucosamine pentamer, (GlcNAc)5. Although GHL was found to retain the main-chain folding similar to HEL as judged from CD spectroscopy, the courses of GHL showed increased production of (GlcNAc)4 and reduced production of (GlcNAc)2 when compared with HEL. To identify critical residue(s) involved in the alteration in the courses of GHL, two mutant enzymes as to subsites E and F in HEL, N113K and R114H, were prepared by site-directed mutagenesis. Kinetic analysis of these mutants revealed that the mutation of Asn113 to Lys had little effect on the courses of HEL, while the Arg114 to His mutation completely reproduced the courses of GHL, demonstrating that His114 in GHL is the key residue responsible for the characteristic courses of GHL. Computer simulation of the reaction courses of the R114H mutant revealed that this substitution decreased not only the binding free energies for subsites E and F, but also the rate constant of transglycosylation. The Arg residue at position 114 may play an important role in the transglycosylation activity of HEL. |
| Databáze: | OpenAIRE |
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