Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2007

Lecture Notes: 22 March

© R. Paselk 2006



Recall the Glycolysis pathway, and note the energy drops in the free energy plot, as seen below:

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.

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 (Lecture 18)

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 17.)

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.

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:

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, the cross-over Energy Charge, as seen in the figure:

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

Pathway Diagrams

C438 Home

Lecture Notes

Last modified 22 March 2007