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Leloir pathway: the crucial route for galactose metabolism and galactosemia

Dr. Nicola Tazzini

The Leloir pathway is the primary route for galactose metabolism.
Discovered by Luis F. Leloir and colleagues in 1948, this pathway converts galactose into glucose 1-phosphate. Specifically, it involves the inversion of the configuration of the hydroxyl group at carbon 4 of galactose, one of the monosaccharide’s chiral centers.[1]

The metabolic intermediates involved in this isomerization serve as building blocks in various biosynthetic pathways, such as the glycosylation of proteins and lipids or glycogen synthesis. Their role depends on the developmental stage, the cell’s metabolic state, and the tissue type.[2]

With the exception of the first step, the reactions in the Leloir pathway are reversible, proceeding in either direction depending on substrate concentrations and the tissue’s metabolic needs. This enables the interconversion of galactose and glucose.[3]

The importance of the Leloir pathway, and of galactose itself, is highlighted by its high degree of evolutionary conservation, from bacteria to plants and animals.[4] In humans, its relevance is further underscored by the severe consequences of mutations in the genes encoding its enzymes, which lead to the inherited metabolic disorder known as galactosemia.[5]

Contents

Galactose

Galactose, along with glucose and fructose, is one of the monosaccharides that can be absorbed in the intestine. The main dietary source of galactose is lactose, which, together with maltose, trehalose and sucrose, is one of the disaccharides commonly found in food.
Since there are no transporters for disaccharides, their glycosidic bonds are hydrolyzed in the final stage of carbohydrate digestion, releasing their constituent monosaccharides. In the case of lactose, these are glucose and galactose.[6]

The monosaccharides are then absorbed and transported via the portal circulation to the liver, the primary site of galactose metabolism. The liver absorbs most of the galactose (approximately 88%) through facilitated passive diffusion mediated by the GLUT2 transporter.[7]

The small remaining amount of circulating galactose reaches other organs and tissues, such as the mammary gland, which during lactation uses galactose for lactose synthesis and for the glycosylation of milk proteins and lipids.[4]

The steps of the Leloir pathway

The Leloir pathway consists of four enzymatic reactions, catalyzed respectively by:

Step 1: galactose mutarotase

The hydrolysis of the β-(1→4) glycosidic bond in lactose results in the release of glucose and β-D-galactose.
Galactokinase, the enzyme responsible for the second step of the Leloir pathway, is specific for the α-anomer of galactose.[8] Therefore, the β-anomer must first be converted into the α-anomer. This conversion is catalyzed by galactose mutarotase.
In addition to galactose, this enzyme is also capable of interconverting the α- and β-anomeric forms of other sugars, including glucose, xylulose, maltose, and lactose, although with varying degrees of efficiency.[9]

Step 2: galactokinase

In the second step, α-D-galactose is phosphorylated to galactose 1-phosphate in a reaction catalyzed by galactokinase.[8] Phosphorylation of galactose, like that of glucose to glucose 6-phosphate, is metabolically important for several reasons.

The reaction catalyzed by galactokinase represents the irreversible step of the Leloir pathway.[3] Unlike hexokinase and glucokinase (EC 2.7.1.1), which phosphorylate the hydroxyl group at carbon 6 of glucose, galactokinase and fructokinase (EC 2.7.1.4) phosphorylate the hydroxyl group at carbon 1 of galactose and fructose, respectively.[12]

The conversion of galactose 1-phosphate to glucose-1-phosphate requires two additional reactions: the third and fourth steps of the Leloir pathway.

Step 3: galactose 1-phosphate uridylyltransferase

In the third step, galactose 1-phosphate uridylyltransferase catalyzes the transfer of a uridine monophosphate (UMP) group from UDP-glucose to galactose 1-phosphate, yielding UDP-galactose and glucose 1-phosphate.
The reaction follows a ping-pong (double displacement) mechanism, involving the formation of a covalent intermediate between the enzyme and the UMP group.[10]

Step 4: UDP-galactose 4-epimerase

In the final step, UDP-galactose is converted into UDP-glucose in a reaction catalyzed by UDP-galactose 4-epimerase. This enzyme inverts the configuration of the hydroxyl group at carbon 4 and is responsible for the interconversion between UDP-galactose and UDP-glucose, and, by extension, between galactose and glucose.[13]

UDP-galactose 4-epimerase requires NAD+ as a cofactor, and the reaction proceeds through the formation of a ketonic intermediate at C4, accompanied by the reduction of NAD+ to NADH. The sugar then rotates within the active site, exposing the opposite face to NADH, which transfers a hydride ion back to carbon 4, but in the reversed configuration.[14]

Because NAD+ is first reduced and then re-oxidized, there is no net redox change for the coenzyme, which therefore does not appear in the overall reaction equation.
UDP-galactose 4-epimerase is thought to be the rate-limiting enzyme of the Leloir pathway.[4]

The UDP-glucose produced in this reaction is subsequently recycled by UDP-glucose pyrophosphorylase, releasing glucose 1-phosphate.[2]
In mammals, UDP-galactose 4-epimerase also catalyzes the interconversion between UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine.[15]

It is important to note that UDP-galactose and UDP-glucose, like galactose and glucose in general, have the same molecular formula. They differ only in the configuration of the hydroxyl group at carbon 4, making them stereoisomers, and more specifically, epimers, a form of optical isomerism.

What is the role of the Leloir pathway?

The Leloir pathway enables cells to utilize galactose or its derivative, glucose, in various metabolic pathways, both anabolic and catabolic, depending on the metabolic state of the cell or tissue.
Because the reaction catalyzed by UDP-galactose 4-epimerase is reversible, the conversion of glucose into galactose (and its nucleotide derivatives) is also possible.[2]
UDP-galactose can be used in:

In the liver and skeletal muscle, UDP-glucose derived from UDP-galactose can be used:[3]

Note: UDP-galactose, discovered during studies on the Leloir pathway, was the first nucleotide sugar to be identified.[19]

Leloir pathway and galactosemia

Glycosylations are post-translational modifications that play a crucial role in enabling and regulating a wide range of biological processes. Defects in glycosylation have been associated with numerous pathological conditions, including cancer, diabetes, and congenital metabolic disorders, particularly the congenital disorders of glycosylation (CDGs), which are primarily autosomal recessive monogenic conditions.[20] Among these, galactosemia is a well-known disorder, first described by von Reuss A. in 1908.[21] Galactosemia results from mutations in one of the genes encoding the enzymes of the Leloir pathway, and four clinical types have been identified.

At present, the standard treatment for all forms of galactosemia consists of a galactose-restricted diet, aimed at preventing the toxic accumulation of galactose or its metabolites.[26]

Galactosemia and cataracts

The accumulation of galactose activates alternative metabolic pathways, including the synthesis of galactitol and galactonate.

One of the early clinical manifestations of galactosemia is the development of cataracts, typically within the first two years of life. In the most severe cases, the disease may also lead to brain, liver, and kidney damage.[9]

One proposed mechanism underlying cataract formation involves the reduction of galactose, accumulated in the lens, to galactitol in a reaction catalyzed by aldose reductase (EC 1.1.1.21).[27] Galactitol is poorly metabolized and does not readily diffuse across cell membranes due to its low lipophilicity. As an osmotically active compound, it increases intracellular osmotic pressure, leading to a net influx of water into the lens cells.[28]

Moreover, its synthesis consumes NADPH, potentially depleting cellular NADPH pools and impairing the activity of glutathione reductase (EC 1.8.1.7). This reduction in antioxidant capacity may contribute to the accumulation of free radicals.[29]

The combined effects of osmotic stress and oxidative damage can ultimately compromise cell integrity, resulting in cell death and the formation of lens opacities. Additionally, galactitol has been reported to act as an inhibitor of galactose mutarotase, which may exacerbate galactose accumulation by further hindering its metabolism.[30]

References

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