Galactokinase is an enzyme (phosphotransferase) that facilitates the phosphorylation of galactose to galactose 1-phosphate at the expense of one molecule of ATP.
Structure
Galactokinase is composed of two domains separated by a large cleft. The two regions are known as the N- and
C-terminal domains, and the
adenine ring of ATP binds in a hydrophobic pocket located at their interface. The
N-terminal domain is marked by five strands of mixed
beta-sheet and five
alpha-helices, and the C-terminal domain is characterized by two layers of anti-parallel beta-sheets and six alpha-helices.
Galactokinase does not belong to the
carbohydrate kinase family, but rather to a class of ATP-dependent enzymes known as the GHMP superfamily.
GHMP is an abbreviation referring to its original members: galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase. Members of the GHMP superfamily have great three-dimensional similarity despite only ten to 20% sequence identity. These enzymes contain three well-conserved motifs (I, II, and III), the second of which is involved in
nucleotide binding and has the sequence
proline-X-X-X-
glycine-
leucine-X-
Serine-Ser-
alanine.
Sugar specificity
Galactokinases across different species display a great diversity of substrate specificities.
E. coli galactokinase can also phosphorylate 2-deoxy-D-galactose, 2-amino-deoxy-D-galactose, 3-deoxy-D-galactose and
fucose. The enzyme cannot tolerate any C-4 modifications, but changes at the C-2 position of D-galactose do not interfere with enzyme function.
Both human and
rat galactokinases are also able to successfully phosphorylate 2-deoxy-D-galactose.
Galactokinase from
S. cerevisiae, on the other hand, is highly specific for D-galactose and cannot phosphorylate
glucose,
mannose,
arabinose, fucose,
lactose,
galactitol, or 2-deoxy-D-galactose.
Moreover, the kinetic properties of galactokinase also differ across species.
The sugar specificity of galactokinases from different sources has been dramatically expanded through directed evolution
and structure-based protein engineering.
The corresponding broadly permissive sugar anomeric kinases serve as a cornerstone for
in vitro and
in vivo glycorandomization.
Mechanism
Recently, the roles of
active site amino acid in human galactokinase have become understood.
aspartic acid-186 abstracts a
proton from C1-OH of α-D-galactose, and the resulting
alkoxide nucleophile attacks the γ-
phosphorus of ATP. A
phosphate group is transferred to the sugar, and Asp-186 may be
deprotonation by
water. Nearby
arginine-37 stabilizes Asp-186 in its
form and has also been proven to be essential to galactokinase function in
point mutation experiments.
Both the aspartic acid and arginine active site residues are highly conserved among galactokinases.
Biological function
The Leloir pathway catalyzes the conversion of galactose to glucose. Galactose is found in
dairy products, as well as in
fruits and
vegetables, and can be produced endogenously in the breakdown of
glycoproteins and
glycolipids. Three enzymes are required in the Leloir pathway: galactokinase, galactose-1-phosphate uridylyltransferase, and UDP-galactose 4-epimerase. Galactokinase catalyzes the first committed step of galactose catabolism, forming galactose 1-phosphate.
Disease relevance
Galactosemia, a rare metabolic disorder characterized by decreased ability to metabolize galactose, can be caused by a mutation in any of the three enzymes in the Leloir pathway.
Galactokinase deficiency, also known as galactosemia type II, is a
recessive gene metabolic disorder caused by a
mutation in human galactokinase. About 20 mutations have been identified that cause galactosemia type II, the main
symptom of which is early onset
cataracts. In lens cells of the human
eye,
aldose reductase converts galactose to
galactitol. As galactose is not being catabolized to glucose due to a galactokinase mutation, galactitol accumulates. This galactitol gradient across the lens cell membrane triggers the
osmosis uptake of water, and the swelling and eventual
apoptosis of lens cells ensues.
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