Left-sided substrate binding of lysozyme: Evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme

• Published in: Biochemistry (Easton) 1992-10, Vol.31 (42), p.10322-10330
• Main Authors: Inoue, Makoto, Yamada, Hidenori, Yasukochi, Takanori, Miki, Takeyoshi, Horiuchi, Tadao, Imoto, Taiji
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Asparagine; Base Sequence; Binding Sites; Biological and medical sciences; Chickens; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Hydrolases; Kinetics; Models, Molecular; Molecular Sequence Data; Muramidase - chemistry; Muramidase - genetics; Muramidase - metabolism; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00157a021

The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast. We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly. Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state. These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex [Pincus, M. R., & Scheraga, H. A. (1979) Macromolecules 12, 633-644]. These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis [Banerjee S. K., Holler, E., Hess, G. P., & Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367].