Glycolysis

As noted above, glycolysis is only the first stage of glucose degradation. Under aerobic conditions, most of the pyruvate formed in glycolysis undergoes complete oxidative degradation to CO₂ and H₂O.

Pyruvate destined for complete degradation is transported to the mitochondria, where it is decarboxylated to acetyl-CoA by pyruvate dehydrogenase. Acetyl-CoA is completely degraded in the citric acid cycle (or tricarboxylic acid cycle; TCA cycle for short). The “H₂” that is produced here is not gaseous but bound to cosubstrates, as NADH + H⁺ and FADH₂, respectively. It is subsequently oxidized in the respiratory chain; it is in this final stage of glucose breakdown that most of the ATP is actually produced.

If glucose is available in excess of immediate needs and glycogen is already stocked up to capacity, it will still be broken down by glycolysis and pyruvate dehydrogenase to acetyl-CoA. However, acetyl-CoA will then not be oxidized, but it will instead be used for fatty acid synthesis; the fatty acids are converted to triacylglycerol. Fatty acid synthesis occurs in the cytosol of cells in the liver and fat tissue.

We will now consider all these pathways in their turn, starting in this chapter with glycolysis.

Glucose occurs in α and β ring forms that are anomeric at the C1 carbon (highlighted). In the presence of water, one form can reversibly change into the other via an open-chain aldehyde form. Polymers of glucose contain either the α or the β ring form. Within most of these polymers, the C1 hydroxyl groups participate in glycosidic bonds, which prevents ring opening, and therefore the spontaneous transitions between the anomeric forms cannot occur. Starch and glycogen consist of α-d-glucose and yield it during degradation, and this form is also the first substrate in glycolysis.

Most reactions that involve the transfer of a phosphate group from ATP to something else are exergonic, that is, they are energetically favorable. For example, the hydrolysis of ATP—that is, the transfer of the phosphate group to water—has a free energy of -35 kJ/mol. It is interesting to note then that ATP is nevertheless quite stable in solution, which means that there must be a high activation energy barrier that resists hydrolysis of the phosphate anhydride bond.

This energy barrier is imposed by the negative charges at the outer sphere of the triphosphate group that shield the phosphorus atoms in the center from nucleophiles which are also negatively charged. Accordingly, to lower this barrier, kinases provide compensating positive charges within their active sites that engage the negative charges on the ATP molecule and thereby clear the way for nucleophilic attack on the phosphorus.

In hexokinase, the negative charges of the phosphate groups are shielded by magnesium ions and by positively charged amino acid side chains in the active site. This facilitates nucleophilic attack by and phosphate group transfer to the substrate.

The charge shielding mechanism solves one problem, but another one remains, namely, how to limit the reaction to the right nucleophile. After all, the most abundant nucleophile in the cell is water, which should make hydrolysis the most likely outcome of ATP activation. This problem is addressed in the next slide.

After binding of its substrate glucose (yellow and red) and its cosubstrate ATP (not shown), hexokinase adopts a closed conformation, in which substrate and cosubstrate are buried within the enzyme, and water is excluded from the active site and from the reaction (left). The magnified view on the right shows the C6 oxygen of glucose peeking out of the enzyme’s active site; ATP would occupy the cavity above. (The structures were rendered from 3b8a.pdb.)

If the enzyme is given xylose instead of glucose, one water molecule can squeeze into the active site along with the sugar. This water molecule assumes the place of the C6 hydroxymethyl group of glucose, and it will be activated by hexokinase to react with ATP, which will result in ATP hydrolysis.

Phosphohexose isomerase provides an excellent example of acid-base catalysis, which involves the reversible protonation and deprotonation of the substrate. The substrate’s hemiacetal bond is opened by the concerted action of a protonated histidine and of a hydroxide ion, which at the outset is bound to a lysine residue. Then, a glutamate residue abstracts a proton from C2 and donates it back to C1, which causes the C=O double bond to migrate from C1 to C2.

Not shown in the picture are several auxiliary charged residues in the active site, which form ion pairs with charges that develop transiently on the substrate, and thereby stabilize the transition state. Closure of the hemiacetal bond in fructose-6-phosphate, which concludes the reaction, is analogous to the initial ring opening and involves the same catalytic amino acid residues [3].

Glyceraldehyde-3-phosphate dehydrogenase provides a straightforward example of covalent catalysis. This reaction mechanism is very common in dehydrogenation reactions, for example in the citric acid cycle and in the β-oxidation of fatty acids. The reaction goes through the following steps:

1. 1.A cysteine in the enzyme’s active site is deprotonated by an auxiliary histidine to a thiolate anion (–S⁻).
2. 2.Glyceraldehyde-3-phosphate (shown as R(=O)–H) binds to the active site, and the thiolate performs a nucleophilic attack on its aldehyde carbon. This yields a tetrahedral intermediate state, in which the substrate becomes bound covalently to the enzyme—hence the term “covalent catalysis”.
3. 3.The covalent intermediate gives up two electrons and two protons to NAD⁺ and the enzyme, which yields NADH and converts the substrate to a thioester. NADH leaves.
4. 4.The thioester is cleaved by a phosphate ion, again through nucleophilic attack. The product (1,3-bisphosphoglycerate) leaves, and the enzyme is restored to its original state.

The redox cosubstrate used by glyceraldehyde-3-phosphate dehydrogenase, nicotinamide adenine dinucleotide (NAD⁺), also accepts most of the hydrogen that accrues in the degradative pathways downstream of glycolysis. Its structure and the details of its reduction by hydrogen are shown in slide 3.3.6.

The panel on the left shows the structure of NAD⁺ and NADP⁺; the redox-active nicotinamide moiety is highlighted. The panel on the right shows the reduction of this moiety by glyceraldehyde-3-dehydrogenase. The electrons and the hydrogen are transferred from the substrate to the C₄ of the nicotinamide. The electrons then redistribute within the ring.

As you can see, NAD⁺ and NADP⁺ differ solely by the absence or presence of a phosphate group at the lower ribose ring, which has absolutely nothing to do with the actual redox chemistry. Why, then, is it there at all? It simply serves as a tag that allows NAD⁺ and NADP⁺ to interact with separate sets of enzymes. The significance of this duality is discussed in a later chapter (see slide 9.3.1).

The mechanism of pyruvate kinase is similar to that of hexokinase—except that the reaction proceeds the other way, producing ATP rather than consuming it. Yet, both reactions are irreversible. How can this be?

In the hexokinase reaction, the terminal phosphate of ATP is converted from an anhydride to a phosphoester, which is inherently lower in energy; this makes the reaction exergonic and irreversible. The same also applies to phosphofructokinase and to most other phosphorylation reactions. However, the pyruvate kinase is a special case. Its intermediate product, which occurs immediately after transfer of the phosphate group from phosphoenolpyruvate to ADP, is enolpyruvate. Removal of the phosphate group allows the enol group to rearrange itself into a keto group. This second step of the reaction is sufficiently exergonic to offset the energetic cost of converting the phosphoester to the anhydride, and it thus pushes the overall equilibrium of the reaction towards ATP formation.

As you know, the most abundant and important energy-rich metabolite in the cell is ATP. Within this molecule, the energy is stored in the energy-rich phosphate anhydride bonds. Cleavage of these bonds is exergonic, and the energy released by cleavage drives the various reactions and processes powered by ATP. Conversely, in creating ATP from ADP, we require energy to form a new phosphate anhydride bond. We just saw how this energy is derived from phosphoenolpyruvate, and we thus may say that the enolphosphate group is another energy-rich group.

The first ATP in glycolysis is formed by cleavage of the carboxyphosphate mixed anhydride in 1,3-bisphosphoglycerate, and we may thus infer that such mixed anhydrides are energy-rich groups, too. The same functional group also occurs in acetylphosphate and in succinylphosphate, and both of these are capable as well to drive the formation of phosphate anhydride bonds in ATP or GTP.

In slide 3.3.5, we saw that the carboxyphosphate was formed from ionic phosphate and a thioester bond. Since the free phosphate ion is low in energy, it follows that the energy that went into the mixed anhydride came from the thioester. This means that thioesters are energy-rich groups as well.

The glyceraldehyde-3-phosphate dehydrogenase reaction (step 6 in slide 3.2) reduces one equivalent of NAD⁺ to NADH. The concentration of NAD⁺ in the cytosol is not high (less than 1 mM), and it must therefore be regenerated from NADH in order for glycolysis to continue. Under aerobic conditions, the hydrogen is transferred from NADH to one of several carriers that deliver it to the respiratory chain in the mitochondria, and ultimately to oxygen. These shuttle mechanisms are discussed in detail in section 6.9. Under anaerobic conditions, this is impossible; therefore, other means for hydrogen disposal are required.

In human metabolism, pyruvate serves as a makeshift hydrogen acceptor under anaerobic conditions; it is reduced to lactate by lactate dehydrogenase (step 11 in slide 3.2). The lactate is released into the bloodstream, where it accumulates; it is removed and recycled after restoration of oxygen supply. The muscle pain caused by lactate accumulation forces us to discontinue anaerobic exercise after a short while.13

Anaerobic glycolysis also occurs in many microbes, which also face the need to reoxidize NADH. Without the option of reverting to oxidative metabolism within a short time span, they must also deal with the continued accumulation of acid. The yeast Saccharomyces cerevisiae solves this problem through ethanolic fermentation: The acid is converted to a neutral and considerably less toxic compound (ethanol) via decarboxylation. The CO₂ developed in this reaction makes bread dough rise up, whereas the ethanol does the same to government tax revenue.14

A typical carrier for passive substrate transport alternates between two conformations that are open to either side of the membrane. On both sides of the membrane, the substrate reversibly binds to the carrier protein; binding and dissociation are governed by mass action kinetics. Transport occurs when the protein changes from the outward-facing to the inward-facing conformation, or vice versa, while a substrate molecule is bound to it.

The sequence of events in passive transport resembles the pattern of Michaelis-Menten enzyme kinetics—in both cases, a solute binds reversibly to a protein, which then performs some state-altering action on it. Accordingly, the velocity of transport by facilitated diffusion follows the familiar Michaelis-Menten law:

where K_M is the Michaelis constant, [S] is the substrate concentration, V_transport is the rate of transport, and V_max is the maximum rate of transport. This rate is reached at high substrate concentrations, that is, when [S] ≫ K_M.

V_max is proportional to the number of carrier molecules. In many tissues, the number of glucose transporters varies depending on insulin levels. In this way, insulin controls the rate of glucose uptake into cells in these tissues (see slide 13.2.16).

At lower substrate concentrations, the rate of transport becomes dependent on K_M, which is inversely related to the affinity of the transporter for the substrate. From equation 3.1, it can be seen that V_transport will be equal to 1/2 V_max when the glucose concentration equals K_M; therefore, the smaller K_M, the more effective the uptake of glucose will be at low concentrations.

The lowest K_M—or in other words, the highest glucose affinity—occurs with the GLUT3 transporter subtype, which is found in the brain. Given that normal plasma glucose levels are between 4 and 7 mM, the brain will extract glucose efficiently at both high and low glucose levels. In organs with GLUT subtypes of lower affinity, the rate of uptake drops more significantly as glucose becomes depleted. The decrease in transport efficiency is most pronounced with the GLUT2 transporter, which occurs in the liver.

Organ-specific variation of glucose affinity is also observed at the stage of glucose phosphorylation. The liver has a special enzyme called glucokinase, which performs the same reaction as does hexokinase but differs from the latter by a higher K_M value.15

Accordingly, in the liver, phosphorylation will proceed more slowly when the level of blood glucose is low, and most of the glucose will be allowed to pass through the liver and make its way into the general circulation. In contrast, at high concentration, the liver will extract a greater share of the available glucose and convert it to glycogen or fatty acids. The kinetic properties of both glucose transport and phosphorylation therefore support the regulatory function of the liver in blood glucose metabolism.

At this point, we must note that the intrinsic kinetic properties of transporter and enzyme molecules are but one piece in the puzzle of glucose regulation. Insulin and several other hormones control the expression, distribution and activity of transporters and enzymes. Hormonal regulation is essential for proper coordination of glucose utilization, as is evident from its severe disturbances in diabetes mellitus and other endocrine diseases (see chapters 13 and 14).

The graphs in this slide and the previous one were plotted using parameters tabulated in references [5–7].