PLANT PHYSIOLOGY , Vol 111, Issue 4 1227-1231, Copyright © 1996 by American Society of Plant Biologists

BIOCHEMISTRY AND ENZYMOLOGY

Specificity of Binding of [beta]-Glucoside Activators of Ryegrass (1->3)-[beta]-Glucan Synthase and the Synthesis of Some Potential Photoaffinity Activators

K. Ng, E. Johnson and B. A. Stone
School of Biochemistry, La Trobe University, Melbourne, Victoria 3083, Australia

Structure-activity relationships among glycoside activators of ryegrass (Lolium multiflorum) (1->3)-[beta]-glucan synthase were investigated using a number of natural and synthetic glycosides, including some carrying photoaffinity functions. There is an absolute requirement for a [beta]-D-glucosyl moiety in the activator, both S- and N-glucosides are active, and the position of the glucosidic linkage in [beta]-glucose disaccharides has a significant effect on the affinity of binding. However, the binding requirement does not extend beyond a single [beta]-D-glucosyl residue, and [beta]-D-oligoglucosides are less effective than disaccharides. The nature of the aglycon has a major influence on the binding affinity. Hydrophobic aglycons lower the concentration required for half-maximal stimulation of the enzyme obtained from an Eadie-Hofstee plot of kinetic data (Ka) for activation, but charged aglycons increase Ka. Relative to methyl-[beta]-D-glucoside and cellobiose (Ka 1.1 mM), the most potent compounds tested were N-[4-(benzoyl)benzoyl]-[beta]-D-glucosylamine and 2[prime]-[4-azidosalicylamino]ethyl-1-thio-[beta]-D-glucoside with Kas of approximately 30 [mu]M. The latter also was tested for its potential to specifically label the [beta]-glucoside-binding site on the synthase, but under the conditions used the binding was found to be nonspecific.