We have looked at the overall pathway of glycolysis (Glycolysis Pathway) and its three phases (Phase 1 [energy invested]: phosphorylation and rearrangement of glucose, Phase 2 [energy repaid]: oxidative phosphorylation of an aldehyde, and Phase 3 [energy profit]: rearrangement of a phospho-ester to an enol-phosphate). Now let's note the energy and kinetic relationships of this pathway as shown in Table I. Note the 
G°' values: some reactions are quite favorable whereas others are unfavorable, but the overall pathway, including triose isomerase, has a net G°' of -44.65 kJ (Glucose to 2 Pyruvates). So Glycolysis is favorable under standard conditions!
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Now look at the K' and Q values: remember that K' gives equilibrium values under standard conditions (with pH = 7), 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 G 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; P-H 15.23)
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?)
- You should expect and predict magnesium as a cofactor in ALL kinases and ALL enzymes using NTPs. (of course there will be exceptions, but you will generally be correct).
- G-6-P inhibits this enzyme (product feedback inhibition), whereas Pi activates.
- This is an expected control strategy for an enzyme producing a pluripotent product/reactant. We need a regulator which indicates the status of multiple uses/needs.
- HK is an excellent example of induced fit, as discussed previously.
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. This base catalyzed reaction sequence interconverts three of the major hexoses, and can be used in understanding some isomerase enzyme mechanisms. The mechanism is symmetrical. You should finish the second half on your own.
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-glu diad 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-glu diad (glu acts to enhance his catalysis much as in catalytic triad of serine proteases, Lecture 19) 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 open, text Figure 14-4, as we saw with the mutarotation of glucose in our study of catalysis; Lecture 18.)
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, 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.
Pathway Diagrams
