Bare-faced curassow lysozyme carrying amino acid substitutions at subsites E and F shows a change in activity against chitooligosaccharide caused by a local conformational change.
A new form of avian lysozyme, bare-faced curassow lysozyme (BCL), was purified and chemically sequenced. Of the 26 substitutions relative to chicken lysozyme, three, F34Y, T47S, and R114H, are of substrate-interacting residues in the E and F subsites, which would contribute to the acceptor binding for transglycosylation. T47S is a novel substitution in this lysozyme class. While other lysozymes also have substitutions at positions 114 and 34, they also contain numerous others, including ones in the other substrate binding sites, A-D. Furthermore, T47S lies on the left side, while F34Y and R114H are located on the right side of the E-F subsites. BCL therefore should allow comparison of the independent contributions of these sites to substrate binding and transglycosylation. The activity toward the N-acetylglucosamine pentamer revealed that the substitutions at the E-F sites reduced the binding free energies at the E-F sites and the rate constant for transglycosylation without the conformation change of other substrate binding sites on the protein. MD simulation analysis of BCL suggested that the substituted amino acids changed the local conformation of this lysozyme at the E-F sites.
Araki
Tomohiro
T
Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404, Japan. taraki@ktmail.ktokai-u.ac.jp
Seki
Shinobu
S
Hirakawa
Hideki
H
Chijiiwa
Yuki
Y
Kawamura
Shunsuke
S
Kuhara
Satoru
S
Torikata
Takao
T
D000117:Acetylglucosamine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001717:Birds; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019295:Computational Biology; D018360:Crystallography, X-Ray; D019281:Dimerization; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D009844:Oligosaccharides; D010455:Peptides; D009113:Muramidase; D014357:Trypsin; D000117:Acetylglucosamine