Galactokinase (PDB ID:1PIE) from Lactococcus lactis
Created by: Arun Dutta
Galactokinase (1PIE) from Lactococcus lactis is a phosphotransferase
that mediates the phosphorylation of α-D-galactose,
a critical process in galactose metabolism.
The kinase family is defined as a group of enzymes designed to transfer
phosphate groups to substrates.
Galactokinase specifically removes a phosphate from adenosine
triphosphate (ATP), a high-energy molecule involved in almost every endergonic cellular
reaction. Lactococcus lactis is a bacterium, but galactokinase is important
in metabolism for a wide variety of organisms, including humans (4). The molecular weight of galactokinase is 46,031
Da and its isoelectric point is 5.13.
Carbohydrate
metabolism is the cell’s primary means of generating ATP. Galactose is a major energy source for many
organisms and its breakdown into high energy molecules is critical for
survival. The Leloir pathway converts D-galactose
to D-glucose 6-phosphate, the starting point for glycolysis. Galactokinase mediates the second step of
this pathway, which is the conversion of α-D-galactose
to D-galactose-1-phosphate. Specifically, galactokinase transfers a phosphate group from ATP to carbon number one of galactose. Galactokinase activity is
ATP-dependent. Although the end goal of
the Leloir pathway is to produce substrates for glycolysis and generate ATP,
there are a few steps along the way that require some energy input from
ATP. Overall, the amount of ATP produced
far exceeds the amount required. The
fact that the galactokinase reaction requires ATP indicates the importance of
this process. The cell would not invest
ATP in a step that doesn’t result in a serious conformational change critical
for continuing metabolism.
Galactokinase
exists as a monomer in cells. It
contains a 13 amino acid α-helix
at its N-terminus immediately followed by 4 β-strands of varying lengths. The two pairs of strands run anti-parallel. Following this section are more α-helices that loop back
and flank the β-sheets. The C-terminal domain is composed of two β-sheets and contains the
active site. One of these β-sheets is a hairpin
motif, which loops back and interacts with the β-sheets in the N-terminus (2).
Galactose and ATP both fit snugly into the active site, which facilitates the transfer of the phosphate group to the sugar. Galactose
binds to galactokinase via a number of residues, specifically the guanidinium group of Arg36 and carboxylate groups of Glu42, Asp45, and Asp183 (1). In addition,
nearby histidine and glycine residues allow for hydrogen-binding with hydroxyl
groups on galactose, strengthening the interaction. The 1-hydroxyl group on the galactose is
destined to be phosphorylated. The
enzyme positions this group about 3.1Å
away from the phosphate group taken from the ATP. The phosphate transfer to the galactose is
quickly completed once the two substrates are positioned correctly.
Galactokinase is
part of the GHMP super-family. The GHMP
super-family are all ATP-dependent kinases with a number a conserved
motifs. One of these motifs
(X-X-X-Gly-Leu-X-Ser-Ser-Ala) is necessary for ATP-binding (2). In galactokinase, this motif is between Pro131 and Ala140 (7). All the GHMP
kinases are small molecules that phosphorylate intermediate metabolites. The DALI server noted a high degree of
structural similarity between galactokinase and mevalonate kinase, an enzyme
that participates in sterol and isoprene synthesis (4).
Another protein
extremely similar to galactokinase is Gal3p, a transcriptional regulator in Sacchromyces cerevisiae. Gal3p is part of the complex of several
proteins that regulates galactokinase expression (9). This complex contains two other proteins:
Gal80p and Gal4p. Gal4p is a
transcriptional activator that upregulates the expression of the various genes
that encode for the proteins involved in the Leloir pathway. Gal80p binds to Gal4p and prevents any
transcriptional activation. This is the
normal configuration of the two proteins.
However, if there are high levels of galactose in the cell, Gal80p
releases Gal4p, which is then free to activate galactokinase and the other
Leloir pathway genes. Gal3p mediates the
destabilization of the Gal80p-Gal4p dimer.
Gal3p is sensitive to galactose and ATP levels in the cell. If there is a rise in galactose levels and plenty of ATP, Gal3p forces Gal80p to release Gal4p, starting the Leloir pathway and advancing metabolism (9).
There is strong evidence to suggest that Gal3p and galactokinase are evolved from each other. The two proteins are 70% identical and 90% similar (8). Both proteins interact with galactose and ATP, but with different end results. While galactokinase catalyzes a reaction between the two, Gal3p is merely a reporter. It’s possible that Gal3p evolved from galactokinase to allow the cell to fine tune its response to its environment, a hallmark of higher organisms and beautiful example of increased complexity of biological systems with evolution. Gal3p has no kinase activity, but cells without Gal3p cannot adapt to changes in their nutrition environment (8). This regulatory system has evolved from the rudimentary enzyme to allow for higher organisms to survive in a wide range of situations.
In humans, galactokinase deficiency is heavily implicated in galactosemia (1). Galactosemia is a disease that arises from the inability to metabolize galactose to glucose 6-phosphate. Type II galactosemia is caused by a buildup of galactose due to deficient galactokinase activity. The cell’s metabolism capabilities are therefore seriously diminished. There a several possible causes for the galactose buildup. It’s possible that the galactokinase gene itself is mutated. Another explanation could be a problem with one of the regulatory molecules discussed previously. Any change to the regulation machinery affects expression of the root enzyme. Symptoms of type II galactokinase include cataract formation and in extreme cases liver damage (5). Most of the defective versions of galactokinase result from point mutations causing improper folding and steric hindrance within the active site. For example, one postulated mutation is the substitution of an Arg for Gly36. The original glycine pokes into a pocket formed by the R-groups of a series of leucines. The increased size of arginine would force a change in backbone conformation, resulting in a misfolded enzyme (1).
Type I and III
galactosemia are caused by the opposite phenomenon: a buildup of
galactose-1-phosphate, the product of galactokinase activity. If other genes further on in the metabolic
pathway are inactive, metabolism cannot continue and galactose-1-phosphate
accumulation rises to toxic levels.
Cases of type I and III galactosemia are usually more severe and can
result in damage to the liver and brain (4).
This makes galactokinase an appealing drug target, although no chemical
has been synthesized as of yet. If a
treatment inhibits galactokinase activity, then galactose-1-phosphate can’t
build up and galactosemia is avoided.
However, this sort of treatment runs the risk of causing type I
galactosemia by increasing galactose levels.
BLAST
analysis of the protein sequence of galactokinase reveals a high degree of
homology between species, underscoring the importance of this enzyme. Galactokinase from Lactococcus lactis shares 92% sequence identity with a homolog in Bacillus coagulans and 90% sequence
identity with Streptococcus pneumonia. Galactose is a major source of energy for all
kinds of organisms, from bacteria up to humans.
The Leloir cycle is critical for deriving energy from this nutrition
source, and therefore galactokinase is highly conserved among similar organisms.
Galactokinase
is small, and it’s a monomer. It’s a
highly specialized machine designed for one thing and one thing only, and
that’s phosphorylating galactose. It’s
not overly complex nor overly bulky.
Galactose and ATP fit snugly in the active site and the phosphate
transfer is completed quickly. It’s a
model of efficiency.