Abstract
An analysis of the acceptor substrate specificity of domain swap mutants of human alpha1,3/4-fucosyltransferases (FucTs) III and V has been carried out. The results demonstrate that changing Asp336 of FucT III to Ala (as in FucT V) produced a protein (III/V1) with a reduced activity with a variety of acceptors. An analysis of the kinetic properties of FucT III and the III/V1 mutant demonstrated that III/V1 had a 40-fold reduction in its affinity for the H-type 1 acceptor substrate (Fucalpha1,2Galbeta1,3GlcNAc) and 4-fold reduction in its affinity for GDP-fucose when compared with FucT III. Further, the overall catalytic efficiency of III/V1 was approximately 100-fold lower than that of FucT III with an H-type 1 acceptor substrate. The complementary domain swap resulting from the change of Ala349 of FucT V to Asp (V/III1) produced a FucT that had higher enzyme activity with a range of acceptor substrates and had a higher affinity for an H-type 2 acceptor substrate (Fucalpha1, 2Galbeta1,4GlcNAc) with an 8-fold higher overall catalytic efficiency than that of FucT V. No significant change occurred in the Km for GDP-fucose for this protein when compared with FucT V. Kinetic parameters of two other FucT domain swaps (III8/V and V8/III), resulting in proteins that differed from FucT III and V at the NH2 terminus of their catalytic domain, were not significantly different from those of the parental enzymes when H-type 1 and H-type 2 acceptor substrates were utilized. Thus, substitution of an acidic amino acid for a nonpolar amino acid (i.e. Asp versus Ala) at the COOH terminus of FucTs produces an enzyme with enhanced enzyme activities. These results, together with the results presented in the accompanying papers (Nguyen, A. T., Holmes, E. H., Whitaker, J. M., Ho, S., Shetterly, S., and Macher, B. A. (1998) J. Biol. Chem. 273, 25244-25249; Sherwood, A. L., Nguyen, A. T., Whitaker, J. M., Macher, B. A., and Holmes, E. H. (1998) J. Biol. Chem. 273, 25256-25260), provide new insights into the structure/function relationships of human alpha1,3/4-FucT enzymes.
Highlights
An analysis of the acceptor substrate specificity of domain swap mutants of human ␣1,3/4-fucosyltransferases (FucTs) III and V has been carried out
Analysis of the enzymatic and substrate specificity properties of these chimeric enzymes indicated they behaved very to both full-length enzymes and the truncated form without the protein A domain [11]. This information, coupled with the ease of purifying the expressed chimeric enzyme via IgG-agarose beads, makes this system very convenient for studying functional aspects of amino acid alterations in the catalytic domain on enzyme activity. In this earlier study [13], we demonstrated that swapping a segment of FucT III, containing eight of the amino acids unique to FucT III, into the analogous location in the FucT V catalytic domain produced a chimeric protein with substantial ␣1,4- as well as ␣1,3-FucT activities
We have prepared three sets of complementary domain swap proteins (Fig. 1) that retain fucosyltransferase activity to investigate the question of which of the amino acid differences that occur between FucT III and V account for their unique substrate specificities
Summary
FucT, fucosyltransferase; type 1 disaccharide, Gal1,3GlcNAc; type 2 disaccharide, Gal1,4GlcNAc; H-type 1, Fuc␣1,2Gal1,3GlcNAc; H-type 2, Fuc␣1,2Gal1,4GlcNAc. amino acid at the COOH terminus of FucTs III and V is important for the binding of H-type 1 and 2 acceptor substrate and their conversion to product
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