glucose + 2 ADP + 2 NAD⁺ + 2 Pi --> 2 Pyruvate + 2 ATP + 2 NADH + 4 H⁺ + 2 H₂O

• Slightly oxidizing Glucose (Glc) to extract a little free energy
• Notice that O₂ is not directly involved; this is an anaerobic process
• 2 ATPs are invested and 4 are produced: net production of 2 ATP
• Dependent on getting energy by substrate-level phosphorylation (as opposed to oxidative phosphorylation), so glucose must be phosphorylated.
• Product is pyruvate

In an anaerobic environment NADH builds up and a lack of NAD⁺ stops glycolysis. The solution is to build a toxic waste dump
pyruvate + NADH --> lactate + NAD⁺ (lactate dehydrogenase)
or
pyruvate --> CO₂ + acetaldehyde (pyruvate decarboxylase)
acetaldehyde + NADH --> ethanol + NAD⁺ (alcohol dehydrogenase)

Both lactate and ethanol are waste products and toxic. Only a little energy in the form of ATP is produced from glucose by glycolysis.

In cells with aerobic lifestyles the citric acid cycle and oxidative phosphorylation are possible. Under this environment, NADH is available to produce more ATP and can be used for anabolic reactions.

Acetyl CoA + P_i + GDP + FAD + 3NAD⁺ -->
   3 NADH + FADH₂ + GTP + CoA + 2 CO₂

A site to permit you to move through glycolysis, step-by-step

Glycolysis

An animation [local] of the steps in glycolysis and links to info about pathology from genetic disorders

Other recommended sites you might visit

• Diagram of pathway [local]
• Chapter on carbohydrate metabolism
• Glycolysis: The Universal Energy Pathway [local]
• Carbohydrate Metabolism [local]

Bioenergetic considerations

The steady-state concentrations of metabolic intermediates in glycolysis are maintained such that 3 steps have strongly negative G values. These steps are catalyzed by hexokinase, phosphofructokinase-1 (PFK-1) and pyruvate kinase. These three steps are strongly regulated. These are the three steps which require new enzymes to reverse the process of glycolysis.

Bioenergetics of Glycolysis (kJ/mol)

Hexokinase: another example of a regulated enzyme

Hexokinase catalyzes the first of the ten reactions in glycolysis. It catalyzes the essential activation of glucose to glucose-6-P. This activation by addition of a covalent phosphate is necessary for reactions involving the addition of glucose moieties (e.g. making glycogen or starch polymers) and for the incorporation of a phosphate that can later be converted to ATP following oxidation of the sugar. The reaction is

D-glucose + ATP <---> D-glucose-6-P + ADP

When you see an enzyme with 'kinase' in its name, this indicates that the enzyme catalyzes the transfer of a phosphate group from ATP to another molecule. The specifics described here pertain to yeast and animals, but not bacteria. Many bacteria have a different system for phosphorylating glucose with phosphoenolpyruvate as it is transported into the cell.

Although this seems to be a simple reaction, it occurs at an important point in the metabolic system, and a system with multiple levels of regulation has evolved. Glucose must be phosphorylated for further metabolism, a process that consumes ATP. Excess phosphorylation is undesirable because it depletes cellular ATP levels and can cause toxic levels of glucose-6-P to accumulate. Metabolism branches after the formation of glucose-6-P. One branch is the catabolic glycolytic pathway that subjects glucose-6-P to a limited oxidation, forming 2 molecules of pyruvate and yielding ATP from substrate-level phosphorylation. The other branch is the conversion of glucose-6-P to glucose-1-P to begin the anabolic process of synthesizing polymeric carbohydrates such as glycogen. The flux of metabolites through these two branches of metabolism are not equal and depend on the metabolic status and needs of the cell.

When cellular energy availability (e.g. ATP or NADH) becomes low, glycolysis is stimulated and glycogen synthesis is inhibited, causing more flux of glucose-6-P through glycolysis; the next enzyme in the pathway is phosphofructokinase. When cellular energy availability is high, glycolysis is inhibited and glycogen synthesis is stimulated, causing more flux of glucose-6-P to glucose-1-P. Thus, the cell needs to avoid excessive formation of glucose-6-P, but needs to maintain a steady-state pool for ready use. This is accomplished by having the hexokinase enzyme inhibited by its product, glucose-6-P. This acts in a fashion similar to the float valve in the tank of a toilet. As the pool of glucose-6-P nears the desired steady-state level, it starts to inhibit the filling of the pool. This ensures that an adequate pool is always ready, but prevents excessive filling of the pool.

The hexokinase molecule evolved from an enzyme with a molecular weight of about 50,000 to one of about 100,000 by a gene duplication. Thus its primary structure is roughly repeated. The enzyme actually contains two sites capable of binding glucose-6-P, the active site in the C-terminal half, and the allosteric site in the N-terminal half which binds the anions glucose-6-P and Pi (Sebastian et al. [1999] Arch. Biochem. Biophys. 362: 203-210). When glucose-6-P, the product of the enzyme's reaction, binds in the allosteric site, the enzyme undergoes a change in conformation that results in a decreased affinity for ATP. This increase in the Km for ATP causes an inhibition of the velocity of the reaction. The affinity of the allosteric site for glucose-6-P is very high (Ki = 10µM) making the enzyme very sensitive to inhibition by the product of the reaction. Inorganic phosphate (Pi) acts as an antagonist to the inhibitory effect of glucose-6-P. An antagonist is something that counteracts or neutralizes the effect of another molecule. When Pi levels increase, they displace glucose-6-P from the hexokinase allosteric site and relieve the inhibition. This signal makes sense because an increase in Pi concentrations is likely indicating that the cellular [ATP] is decreasing and there is a need for an increased flux of metabolites through glycolysis.

It has also been found that the hexokinase protein is subject to covalent modification. The protein is phosphorylated on two different serine residues by protein kinases. Hexokinase is thought to participate in reversible oligomerization of subunits and binding to the membranes in the mitochondrion in response to physiological signals. This oligomerization is thought to decrease the affinity for glucose and slow the reaction. There has been speculation that phosphorylation of hexokinase decreases the degree of oligomerization and may stimulate the activity (Behlke et al. [1998] Biochemistry 37: 11989-11995). However, more research will be needed to understand if this phosphorylation is physiologically significant.

The above mechanisms are called intrinsic controls, because they are internal to the cell. Hexokinase is also subject to extrinsic control by hormones communicating conditions outside of the cell. Insulin is a hormone produced by the pancreas that communicates a situation of high blood sugar to the body, causing an increase in the uptake of glucose by cells. Insulin also signals the change in the levels of two enzymes. The gene coding for phosphoenolpyruvate carboxykinase, a key element in gluconeogenesis, is down regulated to slow this pathway. The same signal stimulates transcription of the gene coding for hexokinase, leading to increased production of the enzyme and increased capacity to phosphorylate glucose (Hall and Granner [1999] J. Basic Clin. Physiol. Pharmacol. 10: 119-133). This is accomplished by the presence of a hormone-regulated response element in the promoter of the hexokinase gene. This is the specific sequence CCACGTCA located on the promoter between the CCAAT box and the TATA box. The hormone response element binds a transcription factor that is stimulated by an insulin-induced signal transduction cascade, causing increased transcription of the hexokinase mRNA.

Finally, there is a level of control provided by a second enzyme, glucokinase, which catalyzes the same reaction as hexokinase. A major difference is that glucokinase has a much higher Km for the glucose substrate. Under conditions of low glucose uptake, most of the glucose phosphorylation is catalyzed by hexokinase because its Km is one-hundredth that of glucokinase. However, when insulin stimulation after a meal causes a large increase in the uptake rate of glucose, the concentrations of glucose within the cell become markedly elevated. Under these conditions, the presence of the second enzyme, glucokinase, permits an increased capacity to phosphorylate glucose and put it into cellular metabolism.

Other sugars must enter the glycolytic pathway to be catabolized.

glycerol + ATP --> glycerol-3-P + ADP (glycerol kinase)

glycerol-3-P + NAD+ --> DHAP + NADH (glycerol-P DH)

In bacteria:

• Fructose + ATP --> Fru-6-P + ADP (fructokinase)
• Mannose + ATP --> Man-6-P + ADP (mannokinase)
• Man-6-P ---> Fru-6-P (phosphomannoseisomerase)
• Fructose + PEP --> Fru-1-P + Pyruvate (PEP phosphotransferase)
• Fru-1-P + ATP --> Fru-1,6-BP + ADP (Fru-1-P kinase)

In liver:

• Fructose + ATP --> Fru-1-P (fructokinase)
• Fru-1-P --> DHAP (dihydroxyacetonephosphate) + glyceraldehyde (Fru-1-P aldolase)
• glyceraldehyde + ATP --> Ga-3-P (triose kinase)

Overview of non-glucose sugar catabolism.

Some sugar metabolic pathways involve nucleoside phosphate derivatives as intermediates:

Galactose + ATP --> Galactose-1-P + ADP (galactokinase)

Galactose-1-P + UDP-Glc --> UDP-Galactose + Glc-1-P (Gal-1-P uridylyltransferase)

UDP-Galactose <--> UDP-glucose (UDP-Galactose-4-epimerase)

Glc-1-P --> Glc-6-P (phosphoglucomutase)