|Lecture Notes: 7 November||
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?)
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 14.7 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.
After a series of unfavorable or marginal reactions now we get a highly favorable reaction again - pulling the pathway forward.
These two reactions couple an oxidation (favorable) to a phosphorylation (unfavorable) to give a substrate level oxidative phosphorylation with the capture of the oxidative energy as ATP.
Note that the energy investment in Stage 1 has now been "paid back" and Glycolysis is now energy neutral. This brings us to the third stage of Glycolysis and our ATP energy "profit."
This enzyme requires 2,3-bis PGA (2,3BPG; DPG) as a cofactor to phosphorylate the enzyme and to maintain the E-P intermediate:
A detailed mechanism for this enzyme is shown below: [overhead]
Note that we need another enzyme to produce the BPG cofactor: Bisphosphoglycerate mutase. This enzyme catalyses the interconversion of 1,3-bis PGA to 2,3-bis PGA, taking a high energy compound to a low energy compound: this enzyme is thus an obvious candidate for control, since if it had much activity it could drain Glycolysis of ATP production! Normally of very lo activity. (But enhanced in RBC's, since they use BPG to control the binding of oxygen by hemoglobin. RBC's also have another enzyme, 2,3-bis PGA Pase to bring BPG back into Glycolysis as 2-PGA, but without making ATP.)
9) Enolase: 2-PGA to PEP
This is an alcohol elimination reaction as you've seen in OChem, with catalysis by Magnesium and using general base catalysis by the enzyme:
Note that a low energy compound (2-PGA, approx 10 kJ) is converted to a high energy compound (PEP, greater than 60 kJ) with very little change in energy overall. Essentially have made the phosphate bond much less stable, while increasing the stability of other bonds in the molecule.
Last modified 10 November 2008