Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2001
Lecture Notes:: 29 October	© R. Paselk 2001
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GLYCOLYSIS 2

We have looked at the overall pathway of glycolysis (Glycolysis Pathway) and its phases. Now let's note the energy and kinetic relationships of this pathway as shown in Table I. Note the DG°' values: some reactions are quite favorable whereas others are unfavorable, but the overall pathway, including triose isomerase, has a net DG°' of -44.65 kJ. So Glycolysis is favorable under standard conditions!

Now look at the K' and Q values: K' remember gives equilibrium values under standard conditions, while Q gives measured values for real tissues. What we want to pay attention to here is differences between these two values (small variations are expected since tissues are not at standard conditions). Here large differences indicate reactions which are not at equilibrium: these reactions must be controlled in some way by the organism! Thus we see large differences for HK, PFK, and PK in brain, and HK and PFK in RBC's. Muscle is like brain (overhead). The DG values are plotted below as well for clarity. Finally the max activity column shows us what kind of flux is possible through these enzymes. (overhead 13.7, MvH)

Figure I. Free Energy changes in rabbit skeletal muscle (Data from Mathews and van Holde, Biochemistry, Benjamin/Cummings (1990))

Now let's look at the individual reactions of Glycolysis.

1) Hexokinase (HK): Glucose to G-6-P.

Here we see a nucleophilic attack by a primary alcohol on the gamma phosphate of ATP (alcoholysis of an acid anhydride). As we would expect this is a very favorable reaction.

Note the involvement of magnesium in this reaction - it is an essential cofactor. (Non-Mg ATP is a potent inhibitor: What kind?) G-6-P inhibits this enzyme (product feedback inhibition), whereas P_i activates.

HK is an excellent example of induced fit, as shown previously when we discussed enzyme specificity (8 October).

The next reaction involves the rearrangement of glucose:

 

2) G-6-P Isomerase: G-6-P to F-6-P.

 

 

 

 

The mechanism here is based on the Lobry-de-Bruyn von Ekenstein mechanism we looked at earlier (28 September)

 

 

Note that this would seem an ideal reaction to catalyze with a general acid/base mechanism. The enzyme has a bell shaped pH profile with pK_a's at 7 & 9 and has his and lys residues in the active site. I suggest that you guess a mechanism based on this information.2) G-6-P Isomerase: G-6-P to F-6-P.

 

 

 

 

3) PhosphoFructoKinase (PFK)-1: F-6-P to F-1,6-bis P.

 

 

 

 

The chemical mechanism here will be the same as for HK. Note the requirement for Magnesium again.

 

PFK is the key regulatory enzyme for Glycolysis: note it regulates the flux into pathway and is the first committed step for Glycolysis.

 

ATP inhibits, giving Sigmoidal kinetics for F-6-P vs. rate. But [ATP] is not important for regulation! (Probably left over from early regulatory system, but under physiological conditions [ATP] doesn't change enough to regulate PFK, by the time [ATP] falls significantly, organism is dead.)

 

AMP releases ATP inhibition, and are important regulators for mammals (lots of phylogenetic variation).

 

Why AMP? [ATP]:[AMP] = approx. 50, while [ATP]:[ADP] = approx. 10. Thus [AMP] changes more and is much more sensitive measure of [ATP] change and thus availability (e.g. a change of about 10% in [ATP] will result in a change of about 400% in [AMP]!). Of course the problem is where does the AMP come from? Turns out there is an enzyme in most tissues catalyzing the interconversion of ATP, ADP and AMP, Adenylate Kinase:

 

 

An important consideration is then to determine a measure of energy in the cell. A common measure, which we will use is Energy Charge (EC):

 

 

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, as seen in the figure:

 

 

Pathway Diagrams

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Last modified 29 October 2001