GALK

Galactokinase is an enzyme facilitating the phosphorylation of α-D-galactose to galactose 1-phosphate at the expense of ATP (adenosine triphosphate). The conversion of β-D-galactose to glucose 1-phosphate is achieved by the action of four enzymes that make up the Leloir pathway. Galactokinase catalyzes the second step of the Leloir pathway, namely the catabolism of β-D-galactose to glucose 1-phosphate, a metabolic pathway found in most organisms. Galactokinase is first isolated from mammalian liver, and has been studied extensively in yeast, archaea, plants, and humans. The enzyme has attracted increasing research attention due to its important metabolic role. Galactokinase defects can result in the diseased state that is referred to as galactosemia. Furthermore, galactokinase-like molecules could act as sensors for the intracellular concentration of galactose and, under suitable conditions, work as transcriptional regulators.

Structure

Galactokinase consists of two domains separated by a large cleft. The two regions are referred as the N- and C-terminal domains, and the adenine ring of ATP binds with a hydrophobic pocket at their interface. The N-terminal domain is characterized by five strands of mixed beta-sheet and five alpha-helices, and the C-terminal domain is marked by two layers of anti-parallel beta-sheets and six alpha-helices. Galactokinase is not a member of the sugar kinase family, but rather belongs to a type of ATP-dependent enzymes known as the GHMP superfamily. GHMP is an abbreviation containing original members: galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase. Members of the GHMP superfamily show great three-dimensional similarity with only 10 to 20% sequence identity and contain three well-conserved motifs (I, II, and III), the second of which is implicated in nucleotide binding and has the sequence of Pro-X-X-X-Gly-Leu-X-Ser-Ser-Ala.

Figure 1. Crystal structure of galactokinase active site from Lactococcus lactis.

Sugar Specificity

Galactokinases from different species demonstrate a great variety of substrate specificities. E. coli galactokinase can perform the phosphorylation of 2-amino-deoxy-D-galactose, 2-deoxy-D-galactose, 3-deoxy-D-galactose and D-fucose. The enzyme shows no tolerance to any C-4 modifications, but changes at the C-2 position of D-galactose do not have impact on enzyme function. Galactokinases in both human and rat are capable of phosphorylating 2-deoxy-D-galactose. On the other hand, galactokinase from S. cerevisiae is highly specific for D-galactose and cannot phosphorylate mannose, glucose, fucose, arabinose, lactose, galactitol, or 2-deoxy-D-galactose. Moreover, the kinetic properties of galactokinase also differ between species. The sugar specificity of galactokinases from different sources has been dramatically expanded through directed evolution and structure-based protein engineering. The corresponding permissive sugar anomeric kinases act as a cornerstone for in vitro and in vivo glycorandomization.

Mechanism

The roles of active site residues in human galactokinase have recently been revealed. Asp-186 abstracts a proton from hydroxyl group at C-1 postion of α-D-galactose, which results in alkoxide nucleophile attacking the γ-phosphorus of ATP. A phosphate group is transferred to the sugar, and then Asp-186 may be deprotonated by water. Nearby Arg-37 could stabilize Asp-186 in its anionic form and has also been evidenced to be necessary to the function of galactokinase. Both the aspartic acid and arginine active site residues are highly conserved among galactokinase.

Biological Function

The Leloir pathway catalyzes the conversion of galactose to glucose. Galactose widely present in dairy products, fruits and vegetables can be produced endogenously in the breakdown of glycoproteins and glycolipids. Galactokinase, galactose-1-phosphate uridylyltransferase, and UDP-galactose 4-epimerase are enzymes essential in the Leloir pathway: Galactokinase catalyzes the first committed step of galactose catabolism, resulting in galactose 1-phosphate.

Disease Implication

Galactosemia, a rare metabolic disorder, is identified by the decreased ability to metabolize galactose, and is caused by an enzyme mutation in the Leloir pathway. Galactokinase deficiency is an autosomal recessive metabolic disorder marked by an accumulation of galactose and galactitol owing to the decreased conversion of galactose to galactose-1-phosphate by galactokinase. The disorder is induced by mutations in the GALK1 gene located on chromosome 17q24. Galactokinase deficiency is one of the three inborn errors of metabolism that could lead to hypergalactosemia, and is inherited as an autosomal recessive trait. Unlike classic galactosemia, galactokinase deficiency does not occur with serious manifestations in early infancy. Its major clinical symptom is the development of cataracts during the first weeks or months of life caused the accumulation of galactitol, which is a product of an alternative route of galactose utilization. It has been suggested that, the dependence on milk consumption of heterozygous carriers with galactokinase deficiency later in life, may lead to presenile cataracts at 20–50 years of age.

Reference

1. Holden H M, Thoden J B, Timson D J, Reece R J. Galactokinase: structure, function and role in type II galactosemia. Cell Mol Life Sci, 2004, 61(19-20):2471-2484.