Effect of carbohydrate and metal ion binding on the reactivity of the essential thiol groups of lima bean lectin.
A free sulfhydryl group previously has been shown to be required for carbohydrate binding to the lectin from lima bean (Phaseolus lunatus) (Gould, N. R. and Scheinberg, S. L. (1970) Arch. Biochem. Biophys. 141, 607-613). Modification of this group by sulfhydryl reagents was specifically inhibited by D-GalNAc. We have further examined the reactivity of sulfhydryl groups in lima bean lectin with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) as a probe for carbohydrate and metal ion binding. The 4 thiol groups in tetrameric lima bean lectin component III gave identical kinetics for reaction with Nbs2 involving formation of a weak noncovalent complex between Nbs2 and the lectin. The pH-independent reactivity of the thiol groups at neutral pH suggested that the thiols may exist as ion pairs with a nearby ionized group. Carbohydrate ligands were competitive inhibitors of thiol modification. The thiol groups on all 4 subunits of lima bean lectin were completely and reversibly protected by carbohydrate binding. The ability of carbohydrates to inhibit thiol modification correlated with their potency as inhibitors in a precipitin inhibition assay. The best inhibitors were the oligosaccharides alpha-D-GalNAc-(1 leads to 3)[alpha-L-fucose-(1 leads to 2)]beta-D-Gal(1 leads to R) and alpha-D-GalNAc-(1 leads to 2)beta-D-Gal(1 leads to R). Apparent thermodynamic parameters for binding of several carbohydrates were determined by measuring the temperature dependence of thiol protection. Removal of the bound metal ions Ca2+ and Mn2+ following dialysis into EDTA inactivated the lectin and increased the reactivity of the thiol groups 60-fold. This conversion was temperature-dependent and could be reversed upon addition of metal ions. The fast-reacting thiol groups were not protected by haptenic sugars from modifications by Nbs2.
D002118:Calcium / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D037121:Plant Lectins; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013696:Temperature
D037102:Lectins; D037121:Plant Lectins; D013438:Sulfhydryl Compounds; C031887:lima bean lectin; D004492:Edetic Acid; D008274:Magnesium; D002118:Calcium