Functional site in alpha-lactalbumin encompasses a region corresponding to a subsite in lysozyme and parts of two adjacent flexible substructures.
Aromatic cluster 1 of alpha-lactalbumin (LA), a substructure adjacent to the cleft, is important for its interaction with galactosyltransferase (GT) and effects on glucose binding in the lactose synthase complex [Grobler, J. A., Wang, M., Pike, A. K., & Brew, K. (1994) J. Biol. Chem. 269, 5106-5114]. The full extent of the functional region in LA has been probed by mutagenesis of residues that are near aromatic cluster 1 or within the cleft that corresponds to the active site in the homologous type c lysozymes. The conserved residues Val42, Gln54, and Ile59, which correspond to residues of lysozyme that act in substrate binding in subsites C to E, together with residues adjacent to aromatic cluster 1, were found to be not required for activity. In contrast, replacing Leu110, a component of the region corresponding to lysozyme subsite F, with His or Glu greatly reduces the affinity of LA for GT while the introduction of Arg lowers the synergism of LA and glucose binding to GT and also reduces the affinity of LA for GT. Substitutions for Ala106, which is adjacent to Leu110 in the structure, also perturb activity. The region of the cleft corresponding to subsite F is important for function in LA as well as in lysozyme since other components of this subsite, His32 and Phe31, are also crucial for LA activity. The qualitatively different effects of various substitutions for Leu110 may be mediated by their influence on His32 or by changes in the structure of the lactose synthase complex.
Malinovskii
V A
VA
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Tian
J
J
Grobler
J A
JA
Brew
K
K
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D018698:Glutamic Acid; D005973:Glutamine; D006639:Histidine; D007532:Isoleucine; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism*; D007930:Leucine; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014633:Valine
D017931:DNA Primers; D007532:Isoleucine; D005973:Glutamine; D018698:Glutamic Acid; D006639:Histidine; D007768:Lactalbumin; D009113:Muramidase; D007930:Leucine; D014633:Valine