Use of site-directed mutagenesis to identify the galactosyltransferase binding sites for UDP-galactose.
Site-directed mutagenesis was utilized to identify binding sites for UDP-galactose in galactosyltransferase (EC 2.4.1.22). Mutant cDNAs were generated by a procedure based on PCR, and the mutated enzymes were expressed in E.coli cells. The mutant enzymes were purified by Ni-NTA Sephadex, and the degree of purification was judged by SDS-PAGE. Purified mutant GTs, F305L, P306V, N307S, N308S, showed dramatic decreases in activities in comparison with the activity of the wild-type GT. Enzyme kinetic analysis revealed that the Km values of F305L, P306V, N307S and N308S for UDP-galactose were, respectively, 9-, 11-, 50- and 20-fold higher than the Km of wild-type GT, but the Km values for manganese were not significantly different from that of the wild-type GT. The quartet mutant F305L/P306V/N307S/N308S showed no activity. From the results of this study it is concluded that amino acids, Phe-305, Pro-306, Asn-307 and Asn-308, in GT are most probably involved in GT catalysis or are located close to the UDP-galactose binding region but are not involved in the binding of manganese.
Zu
H
H
Department of Surgery, Medical College of Ohio, Toledo 43699.
Fukuda
M N
MN
Wong
S S
SS
Wang
Y
Y
Liu
Z
Z
Tang
Q
Q
Appert
H E
HE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D007700:Kinetics; D007788:Lactose Synthase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014531:Uridine Diphosphate Galactose; D007788:Lactose Synthase