Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:

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Phosphofructokinase Regulation I, cont.

Recall that PFK is the key regulatory enzyme for Glycolysis: It regulates the flux into pathway (flux generating step) and is the first committed step for Glycolysis, and [AMP] is the primary regulator as a positive effector. Look at kinetics plots of PFK activity vs. [ATP] and [F-6-P] [Figure 11.15, Chap 11 p 353]

Last time we noted the the main product of Glycolysis is ATP (energy,) so an important consideration is to determine a measure of energy in the cell. A common measure, which we will use, is Energy Charge (EC):

mathematical expression for energy charge

Most cells maintain EC at a constant value with very little variation: as EC drops, catabolic, energy producing pathways, such as Glycolysis, increase in rate, while anabolic, energy consuming pathways decrease in rate. The opposite occurs as EC increases, resulting in a tight control around an optimal value, the cross-over Energy Charge, as seen in the figure:

Plot of energy charge vs. the relative rates of anabolism and catabolism

Irreversible Reactions

Note that HK and PFK, our two regulatory enzymes thus far, 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.

Structural diagram for aldolase reaction

This reaction is an aldol cleavage, the reverse of the aldol condensation discussed in organic chemistry:

Structural diagram for aldol cleavage mechanism

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":

Structural diagram for alpha carbon resonance

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: [Figure 11.5]

Structural diagram for the molecular mechanism of type 1 aldolases

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.

Recall multisubstrate kinetics:

Kinetic Mechanisms

ordered sequential Bi Bi Kinetic mechanism diagram

Ordered Sequential Bi Bi

Random Sequential Bi Bi Kinetic mechanism diagram

Random Sequential Bi Bi

Ping Pong Bi Bi Kinetic mechanism diagram

Ping pong Bi Bi

 

The enzyme shows an Ordered Sequential Uni Bi kinetic mechanism:

Ordered Sequential Uni Bi Kinetic mechanism diagram for aldolase

Phase II Glycolysis Reactions

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

Structural diagram for triose phosphate isomerase reaction

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?) [Figure 6.8]

6) Glyceraldehyde 3-Phosphate Dehydrogenase: GA-3-P to 1,3-bis PGA

Structural diagram for glyceraldehyde 3-phosphate dehydrogenase reaction

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

Cleland diagram for the Ordered Sequential Ter Bi kinetic mechanism of  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:

simplified molecular mechanism for Ga-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 11.7 in your text): [overhead]

Structural diagram for the molecular mechanism of 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 equation for 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."

Phase III Glycolysis Reactions

8) Phosphoglycerate Mutase: 3-PGA to 2-PGA

This reaction begins the third stage of Glycolysis and our ATP energy "profit."

Structural equation for PGA 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 equilibrium arrows 2,3-bisPGA + E equilibrium arrows 2-PGA + E-P

A detailed mechanism for this enzyme is shown below: [Figure 11.8]

Molecular 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 equation for 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:

Molecular 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 15 March 2013