Different reactivity of two brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAc alpha 2-3Gal beta 1-3GalNAc alpha(2-6)sialyltransferase.
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 by N-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialyltransferases. 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 site of the fetuin sialyltransferase (but we could not excluded that CMP-NeuAc 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.
Baubichon-Cortay
H
H
Department of General and Medical Biochemistry, University of Lyon, I.N.S.E.R.M. 198, Oullins, France.
Broquet
P
P
George
P
P
Louisot
P
P
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D001974:Bromosuccinimide / Q000494:pharmacology; D002240:Carbohydrate Sequence; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism* / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D060748:Fetuins; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D000509:alpha-Fetoproteins / Q000378:metabolism; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D001212:Asialoglycoproteins; D060748:Fetuins; D013439:Sulfhydryl Reagents; D000509:alpha-Fetoproteins; C017029:asialofetuin; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; C076584:(Alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl-galactosaminide 6-alpha-sialyltransferase; D001974:Bromosuccinimide; D005033:Ethylmaleimide; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase