Humboldt State University ® Department of Chemistry

Richard A. Paselk
Chem 431

Biochemistry

Fall 2001
Lecture Notes:: 9 November

© R. Paselk 2001
  
     
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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.
 
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). 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.
 
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 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:
 
 
This first part of the reaction is very similar to the first part of the Pyruvate DH catalyzed reaction in the Pyruvate DH Complex which we will look at below. (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:
 
 
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:
 
 
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]

 

 

PYRUVATE METABOLISM

Let's return now to the fate of pyruvate in aerobic tissues. Pyruvate must first be transported into the mitochondria, where it can then be oxidized to give acetyl CoA, which can then be used to make fat for storage or it can be further oxidized to carbon dioxide via the Kreb's TCA Cycle.
 
The oxidation of pyruvate to acetyl CoA is accomplished by the Pyruvate Dehydrogenase complex, a large, multi-component enzyme with three main enzyme subunits. The reactions of the Pyruvate DH Complex are outlined in the diagram.
 
The first enzyme of this complex, pyruvate dehydrogenase (note that, unusual for the DH appellation, there is no direct NAD+ or FAD involvement), catalyzes two sequential reactions. In the first reaction, catalyzed by the alpha subunit of the enzyme, the coenzyme Thiamine Pyrophosphate (TPP), with a highly acidic carbon (a stable carbanion), attacks pyruvate at C-2 with the loss of carbon dioxide to give a covalent coenzyme-substrate intermediate. In the second reaction, catalyzed by the beta subunit, the ketol group is oxidatively transferred to one of the sulfurs of the lipoyl coenzyme on the second enzyme of the complex, dihydrolipoyl transacetylase, to give an acetyl-lipoamide intermediate.
 
The lipoamide of dihydrolipoyl transacetylase constitutes a long arm which may now move the acetyl group from the active site of pyruvate DH to its own active site where the lipoamide is exchanged for Coenzyme A-SH. (On the mammalian enzyme the 60 subunits of the transacetylase seem to form a pool of lipoyl groups among which the acetyl groups are freely exchanged.)
 
 
 
Note that in the reactions of dihydrolipoyl transacetylase the lipoamide has been reduced from a disulfide to two sulfhydryl groups. In order to continue operation lipoamide must be reoxidized and that is accomplished by the final enzyme of the complex, dihydrolipoyl dehydrogenase. The reactions catalyzed by this enzyme are complex, but the net result is the transfer of two electrons from the lipoamide to NAD+ to give NADH. {overhead 14.9, MvH - swinging lipoamide}
 
Overall then the Pyruvate DH Complex converts pyruvate into acetyl CoA in a physiologically irreversible reaction with the release of carbon dioxide and the capture of an electron pair as a hydride ion on NADH. Note the cofactors involved for this reaction sequence: TPP, FAD, Mg2+, lipoamide, Coenzyme A, and NAD+.
 
Structure of Pyruvate DH Complex from bovine kidney: MW = 7 x 106 (without associated Phosphatase)
  1. Transacetylase: 60 subs x 52,000 = 3.1 x 106 arranged as a pentagonal dodecahedron
  2. Dihydrolipoyl DH: 5 dimers x 110,000 = 5 x 105 on faces of dodecahedron
  3. Pyruvate DH: 10 tetramers x 154,000 = 1.54 x 106 on edges of dodecahedron
Pyruvate DH Complex of E. coli is regulated by phosphorylation/dephosphorylation:
 
 
Thus the presence of excess immediate product (AcCoA) or excess ultimate product (reducing equivalents as NADH)shut down the enzyme, while substrate (pyruvate, activates it).

 

KREB'S TCA CYCLE

The Tricarboxylic acid cycle is in many ways the central pathway of metabolism, both catabolically and anabolically: it is involved in the breakdown and synthesis of a variety of compounds. Right now we want to focus of its catabolic role in aerobic catabolism: the oxidative breakdown of the acetyl group of acetyl CoA. In this instance we can consider the entire cycle to be a catalyst for this breakdown.
 
The problem is that the C-C bond of the acetyl group is chemically very resistant. Recall that in organic chemistry generally get C-C bond cleavages at a-b bonds to carbonyl carbons, but with acetyl group there is no beta carbon. So the TCA Cycle creates an a-b bond by first attaching the acetyl group onto a carrier molecule, oxaloacetate.
 

Let's look at an overview of the Kreb's TCA Cycle. First condense the acetyl group with a four carbon carrier to get a six carbon tri-acid. This is then rearranged and oxidized with loss of carbon dioxide to give a five carbon di-acid ketol very similar to pyruvate in structure. An irreversible DH Complex then creates a four carbon CoA derivative with the release of a second carbon dioxide. A series of reactions then regenerates the original carrier.

Pathway Diagrams

 

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Last modified 9 November 2001