To understand the requirement of preincubation of betaine-type analogues with enzymes and/or substrates to activate enzyme reactions, we compared alpha-glucosidase-catalyzed hydrolysis using the analogue 2-(N,N,N-tri-n-butylammonium)acetate and different mixing protocols. We found that enzyme activation is independent of the mixing protocol used and activation initiates immediately following the addition of the analogue to the reaction. Further analysis revealed that facilitation, ascribed to the temporal stabilization of alpha-glucosidase, is primarily applicable to the active enzyme and not to the denatured protein.