Exchange experiments aldolase

Rose et al. [54] have demonstrated by isotope exchange experiments that aldolase operates with an ordered mechanism, as outlined in Scheme 7, in which the formation of an iminium ion with DHAP is followed by the abstraction at C-3 of the pro-S proton. Only after this event may G-3-P bind in a productive manner. A condensation step then can occur to produce the iminium ion of the fructose. In the reverse reaction the cleavage step of the fructose creates an eneamine, but that eneamine may not be protonated before the G-3-P has left the enzyme surface. Any acceptable proposed mechanism for aldolase must include this order of addition of substrates. 

It should be pointed out that the assumption that Schiff base formation should inevitably lead to exchange of solvent [ 0] during the course of an enzymatic reaction has been questioned. For many years, solvent [ 0] exchange experiments were used to distinguish between type I aldolases, which operate via formation of a Schiff base intermediate that links the sub-... 

Stereospecifidly of Dihydroxyacetone Phosphate Reactions. In both the aldolase and triose phosphate isomerase reactions a carbon-hydrogen bond of the hydroxymethyl group is broken. Isotope exchange experiments with tritium have shown that both enzymes catalyze an equilibration between one hydrogen of the substrate and the hydrogen of water. ° The two enzymes do not attack the same hydrogen atom each is specific for only one position. In the projection shown (XII),... 

Representatives of all kinds have been explored for synthetic applications while mechanistic investigations were mainly focussed on the distinct FruA enzymes isolated from rabbit muscle [196] and yeast [197,198]. For mechanistic reasons, all DHAP aldolases appear to be highly specific for the donor component DHAP [199], and only a few isosteric replacements of the ester oxygen for sulfur (46), nitrogen (47), or methylene carbon (48) were found to be tolerable in preparative experiments (Fig. 7) [200,201], Earlier assay results [202] that had indicated activity also for a racemic methyl-branched DHAP analog 53 are now considered to be artefactual [203]. Dihydroxyacetone sulfate 50 has been shown to be covalently bound via Schiff base formation, but apparently no a-deprotonation occurred as neither H/D-exchange nor C-C... 

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