Irreversible Reactions: Note that HK and PFK both catalyze biologically irreversible reactions. That is the enzymes are designed such that the concentrations of the products are far below the KM values under physiological conditions, so the reverse reactions are not catalyzed!
The next reaction involves an almost symmetrical cleavage of F-1,6-bisP to begin phase II:
4) Aldolase: F-1,6-bis P to Glyceraldehyde-3-Phosphate & Dihydroxyacetone Phosphate.
This reaction is an aldol cleavage, the reverse of the aldol condensation discussed in organic chemistry:
Recall that this reaction, where C-C bond making or breaking takes place, is only possible because of the acidity of C-2 (the alpha C), which allows the formation of the nucleophilic carbanion. This acidity can be explained by the resonance structures which may be drawn for the alkyl-carbonyl "group":
Thus in thinking about a catalytic mechanism for this reaction we should look for ways to further stabilize the carbanion, making it an even better leaving group, and therefore making the transition state easier to achieve.
The molecular mechanism for this enzyme, a type I aldolase, is shown below:
Note that the enzyme works on the open form of the sugar, and uses a protonated schiff base intermediate at the heart of the mechanism.
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The enzyme shows an Ordered Sequential Uni Bi kinetic mechanism:
5) Triose Phosphate Isomerase: DHAP to GA-3-P
DHAP is more stable, so most of the aldolase product ends up in the DHAP pool in the cell. Need a high activity enzyme to assure the availability of this pool for proceeding through Glycolysis.
TPI turns out to have a very high turnover number (number of molecules processed per active site per time): approx. 1,000,000 mol/min/site, apparently diffusion controlled. That is this enzyme appears to operate as fast as physically possible: as soon as substrate arrives it is converted. Sometimes referred to as a "perfect catalyst."
As with G-6-P Isomerase it uses a LBE type mechanism with enediol intermediate. Again see 2 pKa's and bell shaped pH titration curve. (What must be different about this mechanism compared to G-6-P Isomerase?)
6) Glyceraldehyde 3-Phosphate Dehydrogenase: GA-3-P to 1,3-bis PGA
GA-3-P DH shows an Ordered Sequential Ter Bi kinetic mechanism:
Oxidizing an aldehyde to an 'acid,' creating a mixed acid anhydride in the process: How? Go through an enzyme bound hemithioacetal which is then oxidized to an enzyme bound high energy thiolester. The thiolester can then be phosphorylized to give 1,3-bis PGA:
Note in this mechanism that the thiol group of cysteine is used both as catalyst and to preserve and transfer the free energy of the oxidation reaction. Thus the carbon of the thiohemiacetal is less (+) than an acetal carbon and so it is easier to remove a hydride ion using NAD+, and the resulting thiol ester is a high energy compound which is readily attacked by phosphate.
Now let's look at the detailed mechanism (A detailed mechanism for this enzyme is also shown as Figure 11.7 in your text): [overhead]
(Note: Arsenate can substitute for phosphate forming highly unstable 1-As-3-PGA, which readily hydrolyses, thus producing no ATP - one mechanism of As toxicity.
Pathway Diagrams
