Study by mutagenesis of the roles of two aromatic clusters of alpha-lactalbumin in aspects of its action in the lactose synthase system.
A new system for the bacterial expression of a variant of bovine alpha-lactalbumin has been developed. Eighteen mutant proteins containing single site substitutions in a cluster of predominantly aromatic residues adjacent to the cleft (aromatic cluster I) and in the hydrophobic box were expressed. The proteins were extracted from inclusion bodies and treated to generate native folding and disulfide bonds to provide appropriately folded protein samples for nine of the mutants. These were characterized with respect to kinetic parameters reflecting aspects of alpha-lactalbumin activity in modulating the specificity of bovine galactosyltransferase. In aromatic cluster I, changes at tryptophan 118 or glutamine 117 were found to specifically reduce affinity for galactosyltransferase, whereas substitutions for phenylalanine 31 or histidine 32 have major effects on the ability to promote glucose binding (13-200-fold) and lesser effects on galactosyltransferase affinity (1.5-70-fold). Substitutions in the hydrophobic box were found to affect folding rather than activity. Thus, the binding of alpha-lactalbumin to galactosyltransferase and its ability to promote glucose binding can be separately perturbed and are associated with distinct but adjacent structures. Aromatic cluster I is directly involved in activity whereas the hydrophobic box appears to have a structural rather than functional role.
Grobler
J A
JA
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Wang
M
M
Pike
A C
AC
Brew
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D017931:DNA Primers; D005700:Galactosyltransferases / Q000378:metabolism; D014644:Genetic Variation; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007788:Lactose Synthase / Q000378:metabolism*; D008433:Mathematics; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D007768:Lactalbumin; D005700:Galactosyltransferases; D007788:Lactose Synthase; D005947:Glucose