What Effects Might Inhibit Glycolysis?

Glycolysis is a universal metabolic process among the world's living things. This series of 10 reactions in the cytoplasm of all cells converts the six-carbon sugar molecule glucose into two molecules of pyruvate, two molecules of ATP and two molecules of NADH.
Learn about glycolysis.

In prokaryotes, which are the simplest organisms, glycolysis is really the only cellular-metabolism game in town. These organisms, almost all of which consist of a single cell with relatively few contents, have limited metabolic needs, and glycolysis is enough to allow them to thrive and reproduce in the absence of competing factors. Eukaryotes, on the other hand, roll out glycolysis as something of a required appetizer before the main dish of aerobic respiration enters the picture.

Discussions of glycolysis often center on the conditions that favor it, e.g., adequate substrate and enzyme concentration. Less often mentioned, but also important, are things that might by design inhibit the rate of glycolysis. Although cells need energy, continually running as much raw material through the glycolysis mill is not always a desired cellular result. Fortunately for the cell, numerous participants in glycolysis have the capacity to affect its speed.

Glucose is a six-carbon sugar with the formula C₆H₁₂O₆. (Fun biomolecule trivia: Every carbohydrate – whether a sugar, a starch or insoluble fiber – has the general chemical formula C_NH_2NO_N.) It has molar mass of 180 g, similar to the heavier amino acids in terms of its size. It is able to diffuse freely into and out of cell through the plasma membrane.

Glucose is a monosaccharide, meaning that it is not made by combining smaller sugars. Fructose is a monosaccharide, while sucrose ("table sugar") is a disaccharide assembled from a glucose molecule and a fructose molecule.

Notably, glucose is in the form of a ring, represented as a hexagon in most diagrams. Five of the six ring atoms are glucose, while the sixth is oxygen. The number-6 carbon lies in a methyl (– CH₃) group outside the ring.

The complete formula for the sum of the 10 reactions of glycolysis is:

C₆H₁₂O₆ + 2 NAD⁺ + 2 Pi + 2 ADP → 2 CH₃(C=O)COOH + 2 ATP + 2 NADH + 2 H⁺

In words, this means that a molecule of glucose is converted to two molecules of glucose, generating 2 ATP and 2 NADH (the reduced form of nicotinamide adenine dinucleotide, a common "electron carrier" in biochemistry).

Note that no oxygen is required. Although pyruvate almost invariably goes on to be consumed in aerobic steps of respiration, glycolysis happens in aerobic and anaerobic organisms alike.

Glycolysis is classically divided into two parts: an "investment phase," which requires 2 ATP (adenosine triphosphate, the "energy currency" of cells) to shape the glucose molecule into something with a great deal of potential energy, and a "payoff" or "harvesting" phase, in which 4 ATP are generated via the conversion of one three-carbon molecule (glyceraldehyde-3-phosphate, or GAP) into another, pyruvate. This means that a total of 4 -2 = 2 ATP are generated per molecule of glucose.

When glucose enters a cell, it is phosphorylated (i.e., has a phosphate group attached to it) under the action of the enzyme hexokinase. This enzyme, or protein catalyst, is among the most important of the regulatory enzymes in glycolysis. Each of the 10 reactions in glycolysis is catalyzed by one enzyme, and that enzyme in turn catalyzes only that one reaction.

The glucose-6-phosphate (G6P) resulting from this phosphorylation step is then converted to fructose-6-phosphate (F6P) before a second phosphorylation occurs, this time at the direction of phosphofructokinase, another critical regulatory enzyme. This results in the formation fructose-1,6-bisphosphate (FBP), and the first phase of glycolysis is complete.

Fructose-1,6-bisphosphate is split into a pair of three-carbon molecules, dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). The DHAP is rapidly converted to GAP, so the net effect of the split is the creation of two identical three-carbon molecules from a single-six carbon molecule.

The GAP is then converted by the enzyme glyceraldehyde-3-phosphate dehydrogenase to 1,3-diphosphoglycerate. This is a busy step; NAD⁺ is converted to NADH and H⁺ using hydrogen atoms stripped from GAP, and then the molecule is phosphorylated.

In the remaining steps, which transform 1,3-diphosphoglycerate into pyruvate, both phosphates are removed in sequence from the three-carbon molecule to generate ATP. Because everything after the splitting of FBP happens twice per glucose molecule, this means that 2 NADH, 2 H⁺ and 4 ATP are generated in the return phase, for a net of 2 NADH, 2 H⁺ and 2 ATP.
Read more about the end result of glycolysis.

Three of the enzymes that participate in glycolysis play major roles in the regulation of the process. Two, hexokinase and phosphofructokinase (or PFK), have been mentioned already. The third, pyruvate kinase, is responsible for catalyzing the final glycolysis reaction, the conversion of phosphoenolpyruvate (PEP) to pyruvate.

Each of these enzymes have activators as well as inhibitors. If you are familiar with chemistry and the concept of feedback inhibition, you might be able to predict the conditions that lead a given enzyme to speed up or slow down its activity. For example, if a region of a cell is rich in G6P , would you expect hexokinase to aggressively seek out any glucose molecules wandering by? You probably wouldn't, because under these conditions, there is no urgent need to generate additional G6P. And you would be correct.

While hexokinase is inhibited by G6P, it is activated by AMP (adenosine monophosphate) and ADP (adenosine diphosphate), as are PFK and pyruvate kinase. This is because higher levels of AMP and ADP generally signify lower levels of ATP, and when ATP is low, the impetus for glycolysis to occur is high.

Pyruvate kinase is also activated by fructose-1,6-bisphosphate, which makes sense, because too much FBP implies that a glycolysis intermediate is accumulating upstream and things need to happen faster at the tail end of the process. Also, fructose-2,6-bisphosphate is an activator of PFK.

Hexokinase, as noted, is inhibited by G6P. PFK and pyruvate kinase are both inhibited by the presence of ATP for the same basic reason they are activated by AMP and ADP: The energy state of the cell favors a decrease in the rate of glycolysis.

PFK is also inhibited by citrate, a component of the Krebs cycle that occurs downstream in aerobic respiration. Pyruvate kinase is inhibited by acetyl CoA, which is the molecule that pyruvate is converted to after glycolysis ends and before the Krebs cycle begins (in fact, acetyl CoA combines with oxaloacetate in the first step of the cycle to create citrate). Finally, the amino acid alanine also inhibits pyruvate kinase.

You might expect other products of glycolysis besides G6P to inhibit hexokinase, since their presence in significant quantities appears to indicate a decreased need for G6P. However, only G6P itself inhibits hexokinase. Why is this?

The reason is fairly simple: G6P is needed for reaction pathways other than glycolysis, including the pentose phosphate shunt and glycogen synthesis. Therefore, if downstream molecules other than G6P could keep hexokinase from its work, these other reaction pathways would also slow down for lack of G6P entering the process, and would therefore represent collateral damage of a sort.