Mechanism of Glucoamylase Reactions: Effect of Urea on the Binding of Maltose and Gluconolactone

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In the presence or absence of urea, binding of a substrate maltose (G2) and a transition-state analogue glucono-1, 5-lactone (GLN) to glucoamylase (GA) from Rhizopus niveus was studied by means of thetransient-phase kinetics using the stopped-flow method. The steady-state kinetics of the GA-catalyzed reactionfor G2 was carried out to evaluate kinetic parameters, Km and k0. Urea is known as a specific reagent for de naturation of proteins, but under the specified conditions of the experiments, we did not find a large denaturation shown by blue-shift in the difference absorption-spectra, indicating that urea-induced change in the conformation of GA was little. The binding kinetics of GLN and G2 agreed well with a two-step mechanism andit was found that urea affects k+2 and k-2 of the two-step mechanism, and Km of the steady-state. These values are closely related with a productive binding of G2 and GLN to subsite 1. Urea is known to effect a weakening of the hydrophobic interaction, so it was suggested that the effect of urea was weakening of the interaction of a saccharide with tryptophan residue(s) located at subsite 1 and in the consequence preventing the formation of a productive complex during the slow unimolecular process.
日本応用糖質科学会の論文