The different enzymes involved in glycolysis act as kinases, mutases, and dehydrogenases, cleaving enzymes, isomerases or enolases. They act in concert to split or rearrange the intermediates, to add on phosphate groups, and to move those phosphate groups onto ADP to make ATP. Several of the reactions involve the phosphorylation of intermediates, which is important not only for the production of ATP from ADP, but also as a useful handle on the substrate for enzyme binding, to trap intermediates within the cell, and to drive pathways in one direction by making phosphorylation and dephosphorylation reactions irreversible. The different enzymes have been split into two groups, those in phase I and those in phase II, simply for convenience.
Catalyses: a-D-Glucose + ATP à Glucose-6-phosphate (G6P) + ADP
The first step in glycolysis is a priming reaction, where a phosphate group is added to glucose using ATP. This reaction is important for its ability to trap glucose within the cell. Whereas glucose can easily traverse the plasma membrane, the negatively charged phosphate group prevents G6P from crossing, so cells can stock up on glucose while levels are high. However, the hexokinase reaction is highly regulated, with G6P providing a feedback inhibition of the enzyme, thereby preventing excessive stockpiling until glycolysis depletes G6P levels.
In mammals, there are four isozymes of hexokinase: types I, II, III and IV (glucokinase). These isozymes differ in their catalysis, localisation and regulation, thereby contributing to the different patterns of glucose metabolism in different tissues. Type I, II and III hexokinases can phosphorylate a variety of hexose sugars, including glucose, fructose and mannose, and as such are involved in a number of metabolic pathways. It is thought that type I hexokinase may have a catabolic function, producing G6P for energy production in glycolysis, whereas types II and III may have an anabolic function, providing G6P for glycogen or lipid synthesis. Type I hexokinase binds to the mitochondrial membrane, thereby enabling the coordination of the rate of glycolysis with that of the TCA cycle. Type IV hexokinase (glucokinase) is a liver/pancreatic b-cell enzyme that is specific for a-D-glucose, and whose level is controlled by insulin, not G6P. Due to the lack of inhibition by G6P, during times of high blood glucose levels the liver can stockpile G6P, converting it to glycogen for later use. In pancreatic b cells, type IV hexokinase acts as a glucose sensor to modify insulin secretion. Mutations in type IV hexokinase have been associated with diabetes mellitus.
Catalyses: Glucose-6-phosphate (G6P) à Fructose-6-phosphate (F6P)
Phosphoglucose isomerase (PGI) catalyses the interconversion of G6P and F6P during glycolysis and gluconeogenesis. The shift of the carbonyl oxygen from the C1 position in G6P to the C2 position in F6P is necessary in order to add another phosphate group at the C1 position in a later reaction.
PGI is a multi-functional enzyme that moonlights as neuroleukin (a neurotrophic factor that mediates the differentiation of neurons), as autocrine motility factor (a tumour-secreted cytokine that regulates cell motility), as differentiation and maturation mediator, and as myofibril-bound serine proteinase inhibitor. Therefore, inside the cell PGI functions in glucose metabolism, while outside the cell it acts as a nerve growth factor and cytokine.
Mutations in PGI are associated with nonspherocytic haemolytic anaemia.
Catalyses: Fructose-6-phosphate (F6P) + ATP à Fructose-1,6-bisphosphate (F1,6PP) + ADP
The third step in glycolysis is another priming reaction, adding a second phosphate group to F6P. This reaction is unidirectional, committing the cell to glycolysis, as opposed to energy storage, or producing a different sugar. A different enzyme, fructose bisphosphatase, is required to catalyse the reverse reaction. The cellular levels of phosphofructokinase (PFK) and fructose bisphosphatase help drive metabolism towards glycolysis or gluconeogenesis, respectively.
PFK is an inducible, highly regulated, allosteric enzyme that is a key regulator of glycolysis. PFK is activated by AMP, ADP, Pi, and fructose-2,6-bisphosphate (F2,6PP), and is inhibited by ATP, citrate, H+ and possibly F1,6PP. In resting muscle, ATP levels are high, while AMP levels are relatively low, contributing to the inhibition of PFK. This inhibition of PFK is reinforced by citrate, an intermediate in the TCA cycle that signals the availability of substrate for aerobic ATP production. By contrast, in working muscle, ATP levels remain fairly constant, while AMP levels rise as ATP and AMP are made from two ADP molecules, signalling the need to activate PFK, and consequently glycolysis. F2,6PP is produced as a metabolic signal, and is not an intermediate in any metabolic pathway. F2,6PP functions as an activator of PFK (glycolysis) and a concomitant inhibitor of fructose bisphosphatase (gluconeogenesis).
Deficiencies in PFK lead to Tauri disease (glycogen storage disease VII), an autosomal recessive disorder characterised by severe nausea, vomiting, muscle cramps and myoglobinuria in response to bursts of intense or vigorous exercise.
Catalyses: Fructose-1,6-bisphosphate (F1,6PP) à dihydroxyacetone phosphate (DHAP) + Glyceraldehyde-3-phosphate (G3P)
Fructose-bisphosphate aldolase (aldolase) catalyses the reversible cleavage of F1,6PP to two triose phosphates, both of which continue through glycolysis.
There are two classes of aldolases, which have different catalytic mechanisms: class I enzymes are found in animals, do not require a metal ion, and are characterised by the formation of a Schiff base intermediate between an active site lysine and a substrate carbonyl group, while the class II enzymes are produced in bacteria and fungi, and require an active-site divalent metal ion. Isozymes are found for each class of enzyme, and in vertebrates the genes encoding each isozyme show tissue-specific expression. For example, class I aldolase A is expressed in muscle, aldolase B in liver, kidney, stomach and intestine, and aldolase C in brain, heart and ovary. The different isozymes have different catalytic functions: aldolases A and C are mainly involved in glycolysis, while aldolase B is involved in both glycolysis and gluconeogenesis. Defects in aldolase B result in hereditary fructose intolerance.
Catalyses: Dihydroxyacetone phosphate (DHAP) à Glyceraldehyde-3-phosphate (G3P)
Triosephosphate isomerase (TIM) catalyses the reversible interconversion of G3P and DHAP. Only G3P can be used in glycolysis, therefore TIM is essential for energy production, allowing two molecules of G3P to be produced for every glucose molecule, thereby doubling the energy yield.
Deficiencies in TIM are associated with haemolytic anaemia coupled with a progressive, severe neurological disorder.
This brings us to the end of the first phase of glycolysis, where a molecule of glucose has produced two molecules of glyceraldehde-3-phosphte, at the cost of two ATP molecules.