Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:

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Glycolysis 3

Phase III Glycolysis Reactions, cont.

Last time we left off with the conversion of 2-PGA to the high energy compound PEP.

9) Enolase: 2-PGA to PEP

Structural equation for Enolase

This is an alcohol elimination reaction as you've seen in OChem, with catalysis by Magnesium and using general base catalysis by the enzyme:

Molecular mechanism for Enolase

Note that a low energy compound (2-PGA, approx 10 kJ) is converted to a high energy compound (PEP, greater than 60 kJ) with very little change in energy overall. Essentially have made the phosphate bond much less stable, while increasing the stability of other bonds in the molecule.

 

10) Pyruvate Kinase: PEP to Pyruvate

Structural equation for Pyruvate kinase reaction

Here we have an attack by ADP:

Molecular mechanism for PK (simplified)

The resulting enol then spontaneously tautomerizes to pyruvate.

FYI - PK Isozymes

PK is a regulatory enzyme in some tissues. There are three isozymes:

  • L-PK (Liver, Kidney, RBC's): greatest regulation. Sigmoidal vs. [PEP] & [K+]; F-1,6-bis P is a (+) effector to both. Hormonal control operates via phosphorylation to inactivate the enzyme.
  • M-PK (Muscle, brain): least regulation - product inhibition by Pyruvate and MgATP.
  • K-PK (Adipose, kidney, liver): intermediate regulation. Sigmoidal vs. [PEP] & [K+]; F-1,6-bis P is a (+) effector to both.

Note how the feed-forward activation of PK by F-1,6-bisP in the liver and adipose(L & K) isozymes along with the feedback inhibition of HK by G-6-P allows PFK to integrate overall regulation of the Glycolysis pathway.

PK completes the reactions of Glycolysis. However, for Glycolysis to proceed NAD+ needs to be regenerated. For aerobic tissues this is done via the Kreb's TCA Cycle. Later we will look at this process for aerobic cells.

Redox Regeneration

Lactate DH: Pyruvate to Lactate

Used to regenerate NAD+ in anaerobic tissue in mammals

Structural equation for Lactate DH reaction

Again the NAD+ abstracts a Hydride ion in the reverse reaction:

Simplified molecular mechanism for Lactate DH

while a general base aids the formation of the carbonyl carbon, and a positive charge draws electron charge up to the carboxyl group and aids the removal of the hydride ion.

If we reverse this reaction to form lactate from pyruvate as in glycolysis, then we see a general acid protonating the carbonyl oxigen to enhance the nucleophilic attack by the NADH Hydride ion on the carbonyl carbon to ruduce pyruvate to lactate.

Lactate DH also has isozymes. It is a tetramer of two types of monomers, H (prevalent in aerobic tissues) & M (prevalent in anaerobic tissues). By random assembly of monomers it has 5 possible isomers: H4, H3M, H2M2, HM3, & M4, with one active site per monomer. The actual distribution of holoenzyme type depends on the relative quantities of monomer synthesized in a given cell type.

FYI - LD Isozymes

Lactate DH is a tetramer of two types of monomers, H & M. The kinetic properties of these pure monomer LD isozymes are given in the Table:

Michaelis Constant (KM)
  H M
Pyruvate 1.4 x 10-4 5.2 x 10-4
Lactate 9 x 10-3 2.5 x 10-2
Pyruvate Inhibition? yes no
The properties of the H(eart) monomer, which predominates in aerobic tissues can be rationalized as better adapted to the aerobic environment. (Heart uses lactate from the serum as a fuel, but doesn't want to lose pyruvate produced in glycolysis to lactate production.

Gluconeogenesis

In order to provide glucose for vital functions such as the metabolism of RBC's and the CNS during periods of fasting (greater than about 8 hrs after food absorption in humans), the body needs a way to synthesis glucose from precursors such as pyruvate and amino acids. This process is referred to as gluconeogenesis. It occurs in the liver and in kidney. Most of Glycolysis can be used in this process since most glycolytic enzymes are operating at equilibrium. However three irreversible enzymes must be bypassed in gluconeogenesis vs. glycolysis: Hexokinase, Phosphofructokinase, and Pyruvate kinase. Phosphofructokinase, and/or hexokinase must also be bypassed in converting other hexoses to glucose.

Pyruvate carboxylase

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:

Diagramatic Pyruvate Carboxylase reaction

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):

Molecular mechanism of part 1 of Pyruvate carboxylate 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:

Molecular mechanism of part 2 of Pyruvate carboxylate reaction

Phosphoenolpyruvate carboxykinase (PEPCK)

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:

Molecular structure mechanism for PEPCK

 

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.

Fructose-bisPhosphatase (F-bisPase)

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 F-bisPase

Glucose-6-Phosphatase (G-6-Pase)

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

Diagram of G-6-Pase reaction

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.

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.

Transketolase

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

Transaldolasecatalyzes 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.

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Last modified 25 March 2013