Chemical modification of an α3-fucosyltransferase; definition of amino acid residues essential for enzyme activity

Biochemical characterization of Silene alba α4-fucosyltransferase and Lewis a products

“…The strong α4-FucT activity in seeds and male flowers suggests that de novo synthesis of Le a -determinants could be associated with androgenesis, and later with seed maturation, as suggested by Joly et al [34]. Use of specific inhibitors showed that Trp and His seem to be key amino acids for S. alba α4-FucT activity as for mammalian FUT3 [35,36] suggesting a possible similar implication of these amino acids in the conserved domain associated with α4-FucT activity. The only slight difference between plant (soluble or membrane anchored enzyme) and FUT3 was found with Cys inhibitor (NEM).…”

“…The only slight difference between plant (soluble or membrane anchored enzyme) and FUT3 was found with Cys inhibitor (NEM). Cys modification by NEM completely inhibited a α3-FucT (FUT6) [35], partially reduced FUT3 [33] activity but had no effect on S. alba α4-FucT. The catalytic domain of FUT3 contains five Cys residues, Cys 143 being responsible for NEM-sensitivity of FUT [33].…”

“…The amino acids Cys, His, Arg, Lys and Trp of human FUT3 and S. alba α4-FucT were modified by NEM, DEPC, phenylglyoxal, 2,4,6-trinitrobenzenesulfonate and N-bromosuccinimide respectively. The recombinant FUT3 used for there experiments was kind gift of Dupuy et al [35]. Enzymes were pre-treated for 30 min with inhibitors prior to measurement of α4-FucT activity.…”

Measurement of α(1-3)Fucosyltransferase Activity Using Scintillation Proximity

“…An identical amount of the FucT-VI preparation (10.5 g) was used in both assays. The sensitivity of FucT-VI (25), along with the lack of sensitivity of FucT-VII (6), to the sulfhydryl reagent N-ethylmaleimide is also maintained (data not shown). Kinetic analysis of FucT-VI also shows the SPA to be comparable with other fucosyltransferase assays.…”

“…␣3 Fucosyltransferase activity was assayed by a modification of Prieels et al (4) as previously described (25). The assay medium contained 100 M GDP-␤-fucose, 100,000 cpm GDP-[ 3 H]fucose, 1 mM ATP, 4 mM MnCl 2 , buffered to pH 7.5 with 50 mM Hepes-NaOH.…”