Abstract
We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited byN-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialytransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl2 did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group, is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.
Similar content being viewed by others
Abbreviations
- p-CMB:
-
p-chloromercuribenzoic acid
- CPDS:
-
6,6′-dithiodinicotinic acid carboxypyridine disulfide
- DTNB:
-
5,5′-dithiobis-(2-nitrobenzoic acid)
- NEM:
-
N-ethylmaleimide
- DTT:
-
dithiothreitol
- Mes:
-
2-(N-morpholino)ethane sulfonic acid
- NeuAc:
-
N-acetylneuraminic acid
References
Schauer R (1982) Cell Biol Monogr 10:205–24.
Powell JT, Brew K (1976) Biochemistry 15:3499–505.
Silvia JS, Ebner KE (1980) J Biol Chem 255:11262–67.
Clymer DJ, Geren CR, Ebner KE (1976) Biochemistry 15:1093–97.
Magee SC, Ebner KE (1974) J Biol Chem 249:6992–98.
Wong LJ, Wong S (1984) Int J Biochem 16:913–17.
O'Keeffe ET, Hill RL, Bell JE (1980) Biochemistry 19:4954–62.
Broquet P, Serres-Guillaumond M, Louisot P (1984) Int J Biochem 16:829–32.
Broquet P, Morelis R, Louisot P (1975) Biochimie 57:983–85.
Baubichon-Cortay H, Serres-Guillaumond M, Louisot P, Broquet P (1986) Carbohydr Res 149:209–23.
Rearick JI, Sadler JE, Paulson JC, Hill RL (1979) J Biol Chem 254:4444–51.
Sadler JE, Rearick JI, Hill RE (1979) J Biol Chem 254:5934–41.
Bergh MLE, Hooghwinkel GJM, van den Eijnden DH (1981) Biochim. Biophys Acta 600:161–69.
Bergh MLE, Koppen PL, van den Eijnden DH (1982) Biochem J 201:411–15.
Bergh MLE, Hooghwinkel GJM, van den Eijnden DH (1983) J Biol Chem 258:7430–36.
Sadler JE, Rearick JI, Paulson JC, Hill RL (1979) J Biol Chem 254:4434–43.
Joziasse DH, Bergh MLE, ter Hart HGJ, Koppen PL, Hooghwinkel GJM, van den Eijnden DH (1985) J Biol Chem 260:4941–51.
Baubichon-Cortay H, Serres-Guillaumond M, Broquet P, Louisot P (1986) Biochim Biophys Acta 862:243–53.
Schaffner W, Weissmann C (1973) Anal Biochem 56:502–14.
Svennerholm L (1957) Biochim Biophys Acta 24:604–11.
Broquet P, Morelis R, Louisot P (1975) J Neurochem 24:989–95.
Mookerjea S, Yung JW (1975) Arch Biochem Biophys 116:223–36.
Levy HM, Leber PD, Ryan EM (1963) J Biol Chem 238:3654–59.
Lee TK, Wong L-JC, Wong SS (1983) J Biol Chem 258:13166–71.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Baubichon-Cortay, H., Broquet, P., George, P. et al. Different reactivity of two Brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAcα2-3Galβ1-3GalNAc α(2–6)sialyltransferase. Glycoconjugate J 6, 115–127 (1989). https://doi.org/10.1007/BF01047894
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01047894