Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431

Biochemistry

Fall 2008

Lecture Notes: 7 November

© R. Paselk 2008
 
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Glycolysis, cont.

5) Triose Phosphate Isomerase (TPI): DHAP to GA-3-P

structural diagram of the reaction catalyzed by triose phosphate isomerase

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

structural diagram of the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase

GA-3-P DH shows an Ordered Sequential Ter Bi kinetic mechanism:

kinetic mechanism diagram for GA-3-P DH

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:

abreviated/dagrammatic mechansm for Glyc 3-P DH

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]

structural diagram of the reaction mechanism for GA-3-P DH

(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.

 

7) Phosphoglycerate Kinase: 1,3-bis PGA to 3-PGA

structural diagram of the reaction catalyzed by phosphoglycerate kinase

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."

8) Phosphoglycerate Mutase: 3-PGA to 2-PGA, begins the third stage of Glycolysis and ATP energy "profit."

structural diagram of the reaction catalyzed by phosphoglycerate mutase

This enzyme requires 2,3-bis PGA (2,3BPG; DPG) as a cofactor to phosphorylate the enzyme and to maintain the E-P intermediate:

3-PGA + E-P double equilibrium arrows 2,3-bisPGA + E double equilibrium arrows 2-PGA + E-P

A detailed mechanism for this enzyme is shown below: [overhead]

structural diagram of the reaction mechanism for PGA mutase

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

structural diagram of the reaction catalyzed by enolase

This is an alcohol elimination reaction as you've seen in OChem, with catalysis by Magnesium and using general base catalysis by the enzyme:

structural diagram of the reaction mechanism for Enolase

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.


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Last modified 10 November 2008