Functional and structural role of a tryptophan generally observed in protein-carbohydrate interaction. TRP-62 of hen egg white lysozyme.
In order to elucidate the role of the aromatic ring in recognition of the sugar ring, Trp-62 of hen egg white lysozyme, which is proposed on the basis of x-ray crystallography data to make contact with a sugar ring through van der Waals interaction, was replaced with aliphatic amino acids (Leu, Ile, Val, and Ala) and Gly by site-directed mutagenesis. In spite of the loss of the aromatic effect, these mutant lysozymes, except for the Trp-62-->Gly mutant, showed higher bacteriolytic activity than the wild-type lysozyme. Furthermore, the Trp-62-->Gly mutant still retained appreciable bacteriolytic activity. On the other hand, by these replacements, the enzymatic activities toward non-charged substrates were markedly reduced. Additionally, the side-chain structure of position 62 was found to be largely responsible for recognition of a saccharide ring in its active site cleft. NMR analysis of the Trp-62-->Leu and Trp-62-->Gly mutants indicated that the structural effects of Trp-62 replacements were localized in the loop region around position 62 and the part of the beta-sheet containing the hydrogen bonding network important for enzymatic activity. Thus, we conclude that Trp-62 not only interacts with oligosaccharide through van der Waals contact, but also maintains the local structural conformation to produce the lysozyme-oligosaccharide interaction.
Maenaka
K
K
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
Kawai
G
G
Watanabe
K
K
Sunada
F
F
Kumagai
I
I
D000818:Animals; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002645:Chickens; D005998:Glycine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry / Q000502:physiology*
D014364:Tryptophan; D009113:Muramidase; D005998:Glycine