Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2007

Lecture Notes: 29 March

© R. Paselk 2006
 
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Gluconeogenesis 2

Let's begin with pyruvate. How is pyruvate converted to PEP without using the pyruvate kinase reaction? Formally, pyruvate is first converted to oxaloacetate, which is in turn converted to PEP. In the first reaction of this process Pyruvate carboxylase adds carbon dioxide to pyruvate with the expenditure of one ATP equivalent of energy. Biotin, a carboxyl-group transfer cofactor in animals, is required by this enzyme:

The reaction takes place in two parts on two different sub-sites on the enzyme. In the first part biotin attacks bicarbonate with a simultaneous attack/hydrolysis by bicarbonate on ATP, resulting in the release of ADP and inorganic phosphate (note the coupling by the enzyme of independent processes in this reaction):

Note that the 14 Angstrom arm of biocytin allows biotin to move between the two sites, in this case carrying the activated carboxyl group. In the second site a pyruvate carbanion then attacks the activated carboxyl group, regenerating the biotin cofactor and releasing oxaloacetate:

Pyruvate carboxylase is followed by the Phosphoenolpyruvate carboxykinase (PEPCK) reaction. In this reaction oxaloacetate is decarboxylated with a simultaneous phosphorylation by GTP to give GDP:

 

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 only 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.

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):

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

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. Examples of two possible balanced gluconeogenesis are shown in the handouts: Gluconeogenesis from Lactate and Gluconeogenesis form Pyruvate. Other systems can also be involved, but we will not look at them.

 

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.

FYI - DH Complex Protein Structure

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

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.

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+.

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 the 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. At this point it appears that acetyl has been released as carbon dioxide, however, the carrier has been reduced, and modified. A series of reactions now regenerates the original carrier.

The first reaction of the cycle is an aldol condensation catalyzed by

Citrate synthase:

Note that the enzyme catalyst enables the coupling of two chemically independent reactions: the aldol condensation (with free energy change of about zero) to the very favorable hydrolysis of the CoA thiol ester bond which drives the overall reaction far towards product. Essentially, we have used an ATP's worth of energy to drive the reaction to completion.
 


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Last modified 29 March 2007