Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431

Biochemistry

Fall 2008

Lecture Notes: 14 November

© R. Paselk 2008
 
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Gluconeogenesis, cont.

The glycolytic reactions from PEP to F-1,6-bisP are fully reversible, but a second bypass is required to get around PFK. This is accomplished by Fructose-bisPhosphatase (F-bisPase):

diagram of the hydrolysis of the 1-phosphate of F-1,6-bis phosphate to F-6-Phosphate

And finally the third irreversible reaction, Glucokinase, is bypassed by Glucose-6-Phosphatase (G-6-Pase):

diagram of the hydrolysis of the  phosphate of G-6-phosphate to Glucose

Note that by hydrolyzing off the phosphates in these two reactions rather than recovering ATP they are made energetically favorable. Thus both glycolysis and gluconeogenesis can be favorable processes, even though they proceed in opposite directions: For Glycolysis, free energy= about - 80 kJ, and for Gluconeogenesis, free energy= about -36 kJ due to differences in ATP. Or another way: if take glucose to pyruvate and then back to glucose again, 4 ATP's are lost.

In order to accomplish gluconeogenesis reducing equivalents in the form of NADH must be provided to the GA-3-P DH enzyme and ATP must be provided to PGA Kinase. The obvious source for these is the Mitochondria, but then there are transport problems. In eukaryotes the transformation of Pyruvate to Phosphoenol pyruvate (PEP) is further complicated by the fact that oxaloacetate is generated from pyruvate and TCA Cycle intermediates in the mitochondria, while PEP is converted to glucose in the cytosol. And oxaloacetate cannot cross the mitochondrial membrane efficiently (it is present at concentrations way below the KM of the carrier, so it must be converted into malate or aspartate in order to cross as summarized in the diagram: gluconeogenesis in the liver. Examples of two possible redox balanced gluconeogenesis paths are shown in the handouts: Gluconeogenesis from Lactate and Gluconeogenesis from Pyruvate. Other systems can also be involved, but we will not look at them.

Sugars other than glucose

Look at gluconeogenesis/glycolysis handout: galactose, mannose, fructose.

Note strategies for each, enzymes, impacts due to entry inyo pathways.

PYRUVATE METABOLISM

Let's turn 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.

Structure of Pyruvate DH Complex from bovine kidney: Text Figure 16-5; MW = 7 x 106 (without associated Phosphatase)

  1. Transacetylase (E2): 60 subs x 52,000 = 3.1 x 106 arranged as a pentagonal dodecahedron
  2. Dihydrolipoyl DH (E1): 5 dimers x 110,000 = 5 x 105 on faces of dodecahedron
  3. Pyruvate DH (E3): 10 tetramers x 154,000 = 1.54 x 106 on edges of dodecahedron

The reactions of the Pyruvate DH Complex are outlined in the diagram. (text Figure 16-6)

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.

structural diagram of the catalytic mechanism of the attack of the TPP carbanion on pyruvate in the enzyme pyruvate DH

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.

structural diagram of the catalytic mechanism of the transfer of the ketol group from TPP to the sulfur on lipoyl in the enzyme dihydrolipoyl transacetylase

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.)

structural diagram of lipoamide (lipoyl group linked via an amide bond to a lysine residue

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.

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+.


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Last modified 14November 2008