Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431

Biochemistry

Fall 2008

Lecture Notes: 7 November

© R. Paselk 2008
 
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Glycolysis, cont.

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

structural diagram of the reaction catalyzed by PFK

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

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:

diagram of equilibriium reaction between 2 ATP and ADP + AMP with magnesium ion as cofactor

Energy Charge

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):

EC = ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP])

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 relative pathways rates vs. energy charge, showing upward growth of anabolic curve and downturn of catabolic curve with increasing EC

 

Irreversible Reactions

Note that HK and PFK 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 of the reaction catalyzed by aldolase

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

structural diagram of the aldol condensation reaction

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 of aldol group 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:

structural diagram of the reaction mechanism for aldolase

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.

The enzyme shows an Ordered Sequential Uni Bi kinetic mechanism as seen in the kinetic mechanism diagram below:

kinetic mechanism diagram for aldolase


Pathway Diagrams

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Last modified 7 November 2008