A highly conserved His-His motif present in alpha1-->3/4fucosyltransferases is required for optimal activity and functions in acceptor binding.
Alpha1-->3/4fucosyltransferases (FucTs) from several species contain a highly conserved His-His motif adjacent to an enzyme region correlating with the ability to catalyze fucose transfer to type 1 chain acceptors. Site-directed mutagenesis has been employed to analyze structure-function relationships of this His-His motif in human FucT-IV. The results indicate that most changes of His(113) and His(114) and nearby residues of FucT-IV reduced the specific activity of the enzymes. Analysis of acceptor properties demonstrated close similarity of most mutants with wild-type FucT-IV, whereas an apparent preference for the H-type II acceptor was observed for the His(114) mutants. Kinetic studies demonstrated that mutants of His(114) had a substantially increased K(m) for acceptor compared to other enzymes tested. The dramatic increase in acceptor K(m) for the His(114) mutants, particularly for the nonfucosylated acceptor, suggests that this His-His motif is involved in acceptor binding and perhaps interacts with GlcNAc residues of type 2 acceptors. The presence of fucose in acceptor substrates may promote more efficient substrate binding and presumably partially overcomes the weaker interaction with GlcNAc caused by the mutation.
Sherwood
Anne L
AL
Northwest Hospital, Molecular Medicine, Department of Cell Surface Biochemistry, 21720 23rd Drive SE, Suite 101, Bothell, WA 98021, USA.
Upchurch
David A
DA
Stroud
Mark R
MR
Davis
William C
WC
Holmes
Eric H
EH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D006639:Histidine; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase