Question

Describe the reaction mechanism for the enzyme aldolase, include the nucleophiles and the electrophiles in the reaction. What would happen to the reaction mechanism if the active site Lysine were mutated to alanine?

Answer 1

To understand the mechanisn of Aldolase catalysed reaction, I am taking example of fructose 1,6-bisphosphate aldolase. The first step in an aldolase reaction is the deprotonation of an alpha-carbon to generate a nucleophilic carbanion Nature has evolved several distinct strategies to stabilize the intermediate that results. Some aldolases use a metal ion to stabilize the negative charge on an enolate intermediate, while others catalyze reactions that proceed through neutral Schiff base or enol intermediates The reaction catalyzed by fructose 1,6-bisphosphate aldolase is a condensation between two 3-carbon sugars, glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP), forming a six-carbon product (which leads, after three more enzymatic steps, to glucose) New C-C bond formed PO PO он Aldolase PO он он dihydroxyacetone phosphate DHAP но OP glyceraldehyde-3-phosphate GAP он fructose 1,6-bisphosphate In the first step of the condensation, an alpha-carbon on DHAP is deprotonated, leading to an enolate intermediate The strategy used to stabilize this key intermediate is to coordinate the negatively-charged enolate oxygen to an enzyme-bound zinc cation OP он он OP Zn Zn Next, the deprotonated a-carbon attacks the carbonyl carbon of GAP in a nucleophilic addition reaction, and protonation of the resulting alcohol leads directly to the fructose 1,6-bisphosphate product.

OPO OH New chiral centre OH OH OP OH OP OH OP GO Zn H A As with many other nucleophilic carbonyl addition reactions, a new stereocenter is created in this reaction, as a planar, achiral carbonyl group is converted to a tetrahedral, chiral alcohol. The enzyme-catalyzed reaction, not surprisingly, is completely stereospecific: the DHAP substrate is positioned in the active site so as to attack the re (front)face of the GAP carbonyl group, leading to the R configuration at the new stereocenter. What would happen to the reaction mechanism if the active site Lysine were mutated to alanine? As we learned all enzyme catalysed reaction are very selective and specific. In this case Lysine is responsible for the activation of OH group, which is driving force for the reaction. If this is mutated by alanine its acivity will change and it will negetively affect the reaction