Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2010

Lecture Notes: 12 April

© R. Paselk 2006
 
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Glycogen Control

Since the Glycogen synthase and phosphorylase reactions are in opposition we need a control system. Glycogen storage/release strategies vary widely with different tissues. In liver, glycogen is used to provide glucose to the serum between meals - it serves a homeostatic function. Glycogen control in liver is thus designed to breakdown and release glycogen when serum [glucose] is low and synthesize glycogen when serum [glucose] is high.

FYI - Glycogen Control in Liver 

In the liver glycogen metabolism is largely regulated by glucose concentrations, which in turn reflect serum glucose concentrations.
  1. In liver glycogen phosphorylase a binds tightly to protein phosphatase-1 and inhibits it. At high concentrations, glucose binds to phosphorylase, causing a release of the protein phosphatase. Protein phosphatase then inactivates m-phosphorylase a by hydrolyzing off Pi to give inactive o-phosphorylase b.
    • Glycogen breakdown and glucose release are inhibited.
  2. Protein phosphatase can now also hydrolyze Pi from inactive Glycogen synthase b to give the active Glycogen synthase a.
    • Glycogen synthesis is activated.
  3. As glucose concentrations drop, glycogen phosphorylase rebinds protein phosphatase and protein kinases rephosphorylate the enzymes, resulting in glucose release.

The net result is that glycogen is synthesized when [glucose] is hi, and it is broken down when [glucose] is low.

Glycogen Control Cascade

In muscle it turns out that glycogen synthesis/breakdown is controlled by a very complex system enabling both rapid response to emergencies and exquisite overall control of the opposing activities to respond to a variety of situations. This is accomplished through the Glycogen Cascade Control system. (The diagram shown is actually a simplified representation, especially of the synthase enzyme, which turns out to have 9 phosphorylatable sites which are phosphorylated by a number of different kinases responding to different complex physiological situations and with varying responses by the enzyme.)

Response begins with a hormonal signal, such as adrenalin, binding to the receptor on the cell surface. This results in the phosphorylation of GDP to GTP on an intracellular G-protein. The G-protein can now interact with Adenylate cyclase to produce the "second messenger" 3', 5'- cyclic AMP (cAMP). Cyclic AMP then binds to the regulatory subunit of cAMP-dependent protein kinase, releasing the active catalytic subunits (C), which can now phosphorylate inactive o-phosphorylase kinase b to the active m-phosphorylase kinase a (o= original, m= modified, b= inactive, a= active). Phosphorylase kinase a then phosphorylates o-Glycogen phosphorylase b to the active m-Glycogen phosphorylase a, resulting in the breakdown of glycogen with the release of G-1-P.

Note the parallel kinase cascade which simultaneously shuts down Glycogen synthase.

Finally, one does not always have a warning, that is time to get the endocrine system going to produce adrenalin, thus the release of Calcium in the muscle cells bypasses much of the cascade, activating the normally inactive o-Phosphorylase kinase b, which then acts on both the phosphorylase and the synthase.

Pentose Phosphate Pathway

The Pentose Phosphate Pathway is an alternate pathway for glucose oxidation which is used to provide reducing equivalents in support of biosynthesis. Thus although it involves the catabolism of glucose, it is generally going to be active only when anabolism is taking place.
This pathway is usually treated in two parts: the oxidative portion, and the sugar interconversions portion. In the oxidative part, on the top of the handout, glucose is first oxidized to a lactone, and then oxidatively decarboxylated. Note that in each case NADP+ is the oxidant as opposed to NAD+. Note also that the two DH reactions are both physiologically irreversible, due in part to the very low concentrations of NADPH in cells.

G-6-P DH

The first enzyme, G-6-P DH, is highly specific for glucose (it is frequently used as the basis of specific glucose assays). In this reaction the #1 (aldehydic) carbon of glucose is oxidized to a lactone (cyclized carboxylic acid ester). This is the first committed step for this pathway and it is regulated by the availability of NADP+ (substrate availability). Since NADP+ and NADPH are in very low concentrations and the NADP+/NADPH ratio is very low, and since NADP+ is generated only during biosynthetic reactions this results in a close coupling of the oxidative portion of this pathway to reductive biosynthesis.

Gluconolactonase

Next Gluconolactonase opens the ring with the addition of a molecule of water. Then 6-P-gluconate DH oxidizes the #3 carbon to a ketone. This results in the #2 carbon becoming somewhat acidic, thus destabilizing the carboxyl group, which is then lost to give the five carbon ribulose-5-P.

In the non-oxidative portion of the Pentose Phosphate Pathway a series of sugar interconversions takes the RU-5-P to intermediates of other pathways: Ribose-5-P for nucleotide biosynthesis, and F-6-P and Ga-3-P for glycolysis/gluconeogenesis. All of these reactions are near equilibrium, with fluxes driven by supply and use of the three intermediates listed above.

In the first two reactions of this phase Ribulose-5-phosphate is converted either to Ribose-5-P via a 1,2-enediol intermediate, or to Xylulose-5-P via a 2,3-enediol intermediate.

These two 5-C sugars, R-5-P and Xu-5-P, are now interconverted to a 7-C sugar, Sedoheptulose-7-P, and a 3-C sugar, Glyceraldehyde-3-P. This reaction is catalyzed by Transketolase, a Thiamine pyrophosphate dependent enzyme which catalyzes the transfer of C2 units. In the first part of this reaction the TPP carbanion (ylid form) makes a nucleophilic attack on the carbonyl group of xylulose. In the resulting intermediate the C2-C3 bond is destabilized and cleavage takes place to yield the enzyme bound 2-(1,2-dihydroxyethyl)-TPP resonance stabilized carbanion:

transketolase mechanism part 1

This first part of the reaction is very similar to the first part of the Pyruvate DH catalyzed reaction in the Pyruvate DH Complex. (Ga-3-P is the leaving group instead of carbon dioxide; there is a 1,2-dihydroxyethyl instead of a 1-hydroxyethyl carbanion intermediate.) In the second part of the reaction the carbanion then attacks the aldehyde of R-5-P to give Su-7-P and regenerate the TPP catalyst:

transketolase mechanism part 2

This is similar to the second part of the Pyruvate DH reaction where the hydroxyethyl group attacks the disufide of the lipoamide. (In this case, of course, the redox catalyzed by the lipoamide does not take place.)

Transaldolase catalyzes the transfer of a C3 unit. The reaction occurs via an aldol cleavage similar to that seen with aldolase: there is a schiff base intermediate formed with an active site lysine. The difference between aldolase and transaldolase is in the acceptor groups: in aldolase the acceptor is a proton, in transaldolase it is another sugar. This reaction yields a F-6-P, which can go to Glycolysis, and an E-4-P which reacts with Xu-5-P catalyzed by the same transketolase seen above. This second transketolase reaction yields F-6-P and Ga-3-P, both intermediates of Glycolysis and the end products of the Pentose-P pathway.

The interconversions of the sugars in this pathway are summarized in the flow diagram below:

Pentose phophate pathway flow diagram

Note that the principle products of this pathway are R-5-P and NADPH. Under reductive biosynthetic conditions where R-5-P is not needed the Pentose-P pathway can be used to completely oxidize G-6-P to 6 carbon dioxide molecules with the concomitant production of 12 NADPH's. Note also that when R-5-P is needed and NADPH is not needed for reductive biosynthesis it can be made from F-6-P and Ga-3-P.

Overview of Glucose Metabolism in the Tissues: Diagram in packet [overhead]

Metabolism of hexoses other than glucose: Looked at Fructose, Mannose and Galactosse on Glyc/Gluconeo Overview.


Pathway Diagrams

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Last modified 4 May 2010