Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2010

Lecture Notes: 29 April

© R. Paselk 2006


Glycolysis 2

Last time we left off with :

Now let's look at the next two phases:

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 DeltaG°' values: some reactions are quite favorable whereas others are unfavorable, but the overall pathway, including triose isomerase, has a net DeltaG°' of -44.65 kJ (Glucose to 2 Pyruvates). So Glycolysis is favorable under standard conditions!

Table I. Free energies, apparent equilibrium constants, mass action ratios, and maximum enzyme activities (in micromol S transformed/min/g fresh tissue) for glycolytic enzymes (Adapted from Newsholme and Start, Regulation in Metabolism, Wiley (1973)).

Glycolytic Enzymes . . Brain . Skeletal Muscle . RBC .
. DeltaG°', kJ K' Q Max Act Q Max Act Q Max Act
Hexokinase -21.94 5000 0.04 17 - 1.5 0.00076 0.3
Hex.Isomerase 2.36 0.4 0.22 80 - 176 0.41 5.6
PFK -17.80 1000 0.13 24 - 56 0.044 1.8
Aldolase 23.73 0.0001 0.000002 15 - 78 0.000014 0.7
Triose Isom. 8.29 0.04 - 415 - 2650 0.35 97
GAP DH - - - 105 - 440 - 17.1
PGA K - - - 610 - 169 - 25.6
DH+K -17.22 800 53 - - - 124 -
Mutase 4.89 0.15 0.1 122 - 100 0.15 8.6
Enolase -3.23 3.5 3.6 47 - 158 1.7 1.6
Pyr K -23.73 10000 5.4 164 - 387 51 4.6
Lac. DH - - - 100 - 366 - 20.4

Now look at the K' and Q values: remember that K' 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 DeltaG values are plotted below as well for clarity. Finally the max activity column shows us what kind of flux is possible through these enzymes - what does this indicate about these tissues and glycolysis?. (overhead 13.7, MvH)


bar chart of glycolysis reaction free energies

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.

Structural diagram for the hexokinase reaction

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.

Structural diagram for the chemical mechanism of the hexokinase reaction

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

Structural diagram for the G-6-P isomerase reaction

The mechanism here is based on the Lobry-de-Bruyn von Ekenstein mechanism we looked at earlier (Lecture 18)

Structural diagram for the Lobry de Bruyn mechnism

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 pKa's at 7 & 9 and has his and lys residues in the active site.

Let's think about this mechanism for a couple of minutes -talk among yourselves and see what you can come up with.

Hexose Isomerase Mechanism: Based on the data provided you should have come up with a mechanism using histidine as a general base catalyst and lysine as general acid catalyst in the first step of the Lobry-de-Bruyn-van Ekenstein Transformation, with a reversal of roles in the second step. (It turns out its more subtle. In fact the lysine is used as a general acid in catalyzing the ring opening as we saw with the mutarotation of glucose in our study of catalysis; Lecture 16.)

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

Structural diagram for the PFK reaction

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

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

Phosphofructokinase Regulation I

PFK is the key regulatory enzyme for Glycolysis: 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 in most organisms. By the time [ATP] falls significantly, organism is dead.)

AMP releases ATP inhibition, and is an important regulator 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:

Structural diagram for the Adenylate kinase reaction

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):

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

The next reaction involves an almost symmetrical cleavage of F-1,6-bisP to begin phase II.

Pathway Diagrams

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Last modified 29 March 2010