Binding kinetics of 4-methylumbelliferyl alpha-mannobioside to concanavalin A by fluorescence stopped-flow measurements.

The stopped-flow binding kinetics of concanavalin A with fluorescently labelled (alpha 1 → 2)-linked mannooligosaccharides are characterized by signal changes which are monophasic for 4-methyl-umbelliferyl alpha-D-mannopyranoside (I), biphasic for 4-methylumbelliferyl alpha-mannobioside(II) and triphasic for 4-methylumbelliferyl alpha-mannotrioside (III). The results indicate that the carbohydrate-binding site interacts specifically with a single mannopyranosyl group, resulting in as many possible complexes as there are mannopyranosyl groups in the oligosaccharide chain. Pseudo-first-order kinetics with II and concanavalin A in the range 6–334 μM yield a faster time constant (110–37 ms) with a saturable amplitude and a slower one (3.9–0.29s) with an amplitude which reaches a peak at 30 μM. The concentration dependencies of the two apparent rate constants and the corresponding amplitudes are compatible with two equivalent mechanisms for which the reaction-rate parameters are calculated. Each involves two complexes with spectroscopic parameters which are dependent upon the mechanism. The first mechanism consists of binding followed by an isomerisation, interpreted as sliding of the internal mannopyranosyl residue to bind preferentially via the terminal residue. In the second mechanism, these two binding modes would alternate; here, the binding via the terminal residue is preferred at equilibrium by a factor of 68; both association-rate parameters are comparable and similar to those for I or simple glycosides. Consistent results were obtained by temperature-jump relaxation kinetics.

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