Complete amino acid sequence of three reptile lysozymes.
To study the structure and function of reptile lysozymes, we have reported their purification, and in this study we have established the amino acid sequence of three egg white lysozymes in soft-shelled turtle eggs (SSTL A and SSTL B from Trionyx sinensis, ASTL from Amyda cartilaginea) by using the rapid peptide mapping method. The established amino acid sequence of SSTL A, SSTL B, and ASTL showed substitutions of 43, 42, and 44 residues respectively when compared with the HEWL (hen egg white lysozyme) sequence. In these reptile lysozymes, SSTL A had one substitution compared with SSTL B (Gly126Asp) and had an N-terminal extra Gly and 11 substitutions compared with ASTL. SSTL B had an N-terminal extra Gly and 10 residues different from ASTL. The sequence of SSTL B was identical to soft-shelled turtle lysozyme from STL (Trionyx sinensis japonicus). The Ile residue at position 93 of ASTL is the first report in all C-type lysozymes. Furthermore, amino acid substitutions (Phe34His, Arg45Tyr, Thr47Arg, and Arg114Tyr) were also found at subsites E and F when compared with HEWL. The time course using N-acetylglucosamine pentamer as a substrate exhibited a reduction of the rate constant of glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution for amino acids mentioned above for substrate binding at subsites E and F. Interestingly, the variable binding free energy values occurred on ASTL, may be contributed from substitutions at outside of subsites E and F.
Ponkham
Pornpimol
P
Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.
Daduang
Sakda
S
Kitimasak
Wachira
W
Krittanai
Chartchai
C
Chokchaichamnankit
Daranee
D
Srisomsap
Chantragan
C
Svasti
Jisnuson
J
Kawamura
Shunsuke
S
Araki
Tomohiro
T
Thammasirirak
Sompong
S
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D004527:Egg Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014426:Turtles / Q000378:metabolism*
D004527:Egg Proteins; C421864:hen egg lysozyme; D009113:Muramidase; D000117:Acetylglucosamine