Thermodynamic and kinetic studies on the mechanism of binding of methylumbelliferyl galactosides to the basic agglutinin from winged bean (Psophocarpus tetragonolobus).
The binding of winged bean basic agglutinin (WBA I) to 4-methylumbelliferyl (MeUmb) galactosides was examined by extrinsic fluorescence titration and stopped-flow spectrofluorimetry. Upon binding to WBA I, MeUmb alpha-galactosides show quenching in fluorescence intensity, decrease in UV absorbance with a concomitant blue shift, and decrease in fluorescence excited-state lifetimes. However, their beta-analogues show enhancement in fluorescence intensity, increase in UV absorbance with a red shift, and an increase in fluorescence excited-state lifetimes. This implies that the umbelliferyl groups of alpha- and beta-galactosides experience non-polar and polar microenvironments, respectively, upon binding to WBA I. Replacement of the anomeric hydroxyl group of galactose by 4-methylumbelliferyl moiety increases the affinity of resulting saccharides. Substitution of C-2 hydroxyl of galactose by an acetamido group leads to increased affinity due to a favorable entropy change. This suggests that acetamido group of MeUmb-alpha/beta-GalNAc binds to a relatively non-polar subsite of WBA I. Most interestingly, this substitution also reduces the association rate constants dramatically. Inspection of the activation parameters reveals that the enthalpy of activation is the limiting factor for the differences in the forward rate constants for these saccharides and the entropic contribution to the activation energy is small.
Puri
K D
KD
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Khan
M I
MI
Gupta
D
D
Surolia
A
A
D000116:Acetylgalactosamine / Q000378:metabolism; D007887:Fabaceae; D005456:Fluorescent Dyes / Q000378:metabolism*; D005697:Galactosides / Q000378:metabolism*; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D037121:Plant Lectins; D010946:Plants, Medicinal; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D005456:Fluorescent Dyes; D005697:Galactosides; D037102:Lectins; D037121:Plant Lectins; C049304:winged bean lectin; D006923:Hymecromone; C026845:4-methylumbelliferyl-galactopyranoside; D000116:Acetylgalactosamine