Carbohydrate Metabolism

Outline

• What happens in glycolysis
• Why it's important
• The ten enzymatic steps
• The fate of pyruvate
• Free energy in glycolysis
• Regulation of glycolysis
• Other sugars in glycolysis
• Bisphosphoglyerate in erythrocytes
• Gluconeogenesis
• Pentose phosphate shunt

What happens in glycolysis

Why it's important

1. Energy in the form of ATP; this is used as fuel for many other reactions.
2. Reducing power in the form of NADH; this is required for oxidation-reduction reactions.
3. Pyruvate, which is a significant starting point both for the Krebs cycle and for lipid biosynthesis.

The ten enzymatic steps

• Hexokinase transfers the γ-phosphoryl group of ATP to the oxygen atom at C-6 of glucose, producing glucose 6-phosphate and ADP. This is an instance where the coupling between ATP hydrolysis and an energy-requiring reaction is very close, because the phosphate is transferred directly from ATP to the recipient molecule, in this case glucose. Most enzymes that carry out a reaction of this kind have the word "kinase" at the end of their name.
  The reaction catalyzed by hexokinase is energetically favored:
  ΔG⁰ ~ -5.33 kcal/mol, so at 310K (human body temperature)
  K_eq = exp(-ΔG⁰/RT)
   = exp(5.33 kcal/;mol / [(1.987 * 10-3 kcal./mol-deg) * 310deg)]
   = exp(5.33/(1.987*0.31)) = 5700, so under conditions of adequate ATP, the equilibrium will definitely favor the product (glucose 6-phosphate) over glucose.
  
  There are various isozymes (functionally related but structurally slightly distinct) forms of hexokinase in humans; the liver form has a K_m in the millimolar range, perhaps a factor of 1000 higher than the K_m of the hexokinase found in other tissues. The liver form is therefore much less active than the other forms unless the liver glucose concentration is high. Hexokinase is active on sugars besides glucose; the activity against maltose is comparable to the activity on glucose. Hexokinase has the highest molecular mass per monomer of any of the glycolytic enzymes; given that it is the first enzyme in an important pathway, it makes sense that it is large and complex.
  
• Phosphoglucomutase  or phosphoglucose isomerase interconverts two phosphorylated forms of glucose--glucose 1-phosphate and glucose 6-phosphate.
  The intermediate is bisphosphorylated, and the equilibrium between the 1-phosphate and 6-phosphate forms is determined by relative concentrations. This enzyme is active on other phosphorylated aldoses in addition to glucose. Note that this enzyme does not appear on the chart above, because it is not part of the linear pathway from glucose to pyruvate.
  
  
  
  
• Phosphohexoseisomerase or glucose 6-phosphate isomerase interconverts two monophosphorylated sugars--glucose 6-phosphate and fructose 6-phosphate.
  As discussed earlier, this interconversion proceeds through a (1,2) ene-diol intermediate; with the enzyme present the energy barriers around this ene-diol are lowered enough to speed the interconversion. This dimeric enzyme plays roles extracellularly as well as intracellularly: it can function as a nerve growth factor. Each monomer contains two unequal-sized domains, and the active site is formed by the association of the two subunits.
  
  
  
  
  
• Phosphofructokinase catalyzes phosphorylation at the 1 position of fructose 6-phosphate.
  It is an example of a kinase that acts on an already-phosphorylated form, creating a bisphosphorylated compound. Of all the enzymes in this pathway it appears to be the one for which the least structural information is available; note that the best structure determined to date was Phil Evans's 2.4 Å structure from 1988, and there have not been many other structures done. ADP acts as an allosteric activator on this enzyme as well as being a product of the reaction.
  
  
  
• Aldolase catalyzes the actual carbon-carbon bond cleavage step.
  This is a large and important enzyme, and structure determinations began (unsuccessfully) more than 25 years ago. Some bacterial and yeast forms require a divalent cation as a cofactor, but the eukaryotic aldolases do not. The non-cationic forms proceed through an imine (Schiff-base) intermediate. The enzyme is active on fructose 1-phosphate as well as its "standard" substrate, fructose 1,6-bisphosphate; in this context it forms part of the catabolic pathway by which fructose itself can be used as an energy and carbon source.
  
  
  
• Triosephosphate isomerase (sometimes written with a space after "triose",
  and for some reason abbreviated "TIM") is possibly the most efficient enzyme known, in terms of the rate acceleration afforded by the enzyme relative to the uncatalyzed reaction. It is a tetrameric enzyme with a characteristic structure in which alpha helical stretches alternate with beta strands such that the beta strands curve around to form a barrel-like structure with the helices outside. This structural motif appears in many other enzymes, and has become known as a "TIM barrel."
  
  
• Glyceraldehyde 3-phosphate dehydrogenase is a medium-sized tetrameric enzyme,
  responsible for the conversion of its substrate to 1,3-bisphosphoglycerate. It resembles several other tetrameric NAD-dependent oxidoreductases, like lactate dehydrogenase, alcohol dehydrogenase and malate dehydrogenase; all have characteristic structures in the NAD-binding region known as "Rossmann folds", after Michael Rossmann, who first characterized this class of enzymes structurally. The enzyme is somewhat allosteric.
  
  
  
  
• Phosphoglycerate kinase catalyzes the dephosphorylation of 1,3-bisphosphoglycerate.
  It is named for the reaction running in the opposite direction relative the one shown in the chart and table above. In the direction shown in the table it produces ATP rather than consuming it. This enzyme has been shown to have a hinge motion about a point near the center of the molecule; the open and closed forms of the enzyme involve movements as large as 17Å in the residues farthest from the hinge point. This enzyme is primarily alpha-helical in conformation.
  
  
  
• Phosphoglycerate mutase interconverts 3-phosphoglycerate and 2-phosphoglycerate.
  According to Mathews's textbook,

  The mechanism of the reaction catalyzed by phosphoglycerate mutase involves formation of 2,3-bisphosphoglycerate via transient phosphorylation of a histidine residue of the enzyme. 2,3BPG can diffuse from phosphoglycerate mutase, however, leaving the enzyme trapped in an unusable state. Cells make excess 2,3BPG (using the enzyme bisphosphoglycerate mutase) in order to drive 2,3BPG back to phosphoglycerate mutase, so the reaction can go to completion.
  

  
  
  
• Enolase converts 2-phosphoglycerate to phosphoenolpyruvate (PEP).
  This reaction plays a role in gluconeogenesis as well as glycolysis. Mg²⁺ ions are required for activity, at least in some forms of the enzyme. Vertebrate genes code for two slightly different forms of the monomer of enolase, alpha and beta. Most of the enolase in fetal tissue is alpha-alpha; mature skeletal muscle contains beta-beta; some alpha-alpha remains in smooth muscle tissue.
  
  
  
• Pyruvate kinase is the last enzyme in the pathway.
  It transfers a phosphate from phosphoenolpyruvate to ADP, producing pyruvate and ATP. The reaction is essentially irreversible. Fructose 1,6-bisphosphate, the substrate for the aldolase reaction, is an activator of this enzyme, affording a level of control, known as "feed-forward activation," over glycolysis. Four isozymes of pyruvate kinase are found in humans, derived from two genes. The fetal (M1) isozyme is nonregulated; the allosterically regulated forms predominant in adults.
  

The fate of pyruvate

Free energy in glycolysis

Examine carefully fig. 11.12 in Horton. The point it makes is that, although the standard free energies associated with the various reactions in glycolysis vary widely, the true free energy changes are monotonically negative and rather small as we go from glucose to pyruvate.In particular, there are really only three steps in the process that are effectively irreversible: the first, third, and last steps, i.e. the hexokinase, phosphofructokinase, and pyruvate kinase steps. All the others have ΔG values close to zero. So the only steps that are irreversible are the ones that involve formation or breakage of high-energy phosphate bonds. The difference between free energy and standard free energy is one we emphasized in the previous chapter. In this instance, the relative abundances of the various metabolites involved in glycolysis drives the reactions whose ΔG^o' values are positive toward the right.

Regulation of glycolysis

This brings up a related point: irreversible reactions tend to be the reactions for which control mechanisms come into play. Horton offers a description of hexose transporters, which are proteins involved in moving hexoses around from one cell to another. There are also control mechanisms that operate by inhibition of specific enzymes in the pathway. In glycolysis, the enzymes on which inhibitory controls are exerted are the three kinase steps discussed above.

Other sugars in glycolysis

Bisphosphoglyerate in erythrocytes

Gluconeogenesis

The pentose phosphate shunt