Histidine-114 at subsites E and F can explain the characteristic enzymatic activity of guinea hen egg-white lysozyme.
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.
Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404, Japan.
Gen
G
Toshima
Shunsuke
S
Kawamura
Tomohiro
T
Araki
Takao
T
Torikata
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002645:Chickens; D004529:Egg White; D006031:Glycosylation; D006639:Histidine / Q000235:genetics* / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D006639:Histidine; D001120:Arginine; C421864:hen egg lysozyme; D009113:Muramidase; D000117:Acetylglucosamine