Effect of amino acid changes in human GALK on their enzymatic properties and the IC50 of selected inhibitors
Site-directed mutagenesis (
2.1. What is galactosemia?
Galactose is a hexose that differs from glucose only by the configuration of the hydroxyl group at the carbon-4 position. Often present as an anomeric mixture of α[?]-D-galactose and β[?]-D-galactose, this monosaccharide exists abundantly in milk, dairy products and many other food types such as fruits and vegetables (Berry, Palmieri et al., 1993; Acosta and Gross, and Gross 1995; Berry et al., 1993). However, galactose can also be produced endogenously in human cells, mainly as products of glycoprotein and glycolipid turnover.(Berry et al., 1995, 2004). Once freely present inside the cells, β[?]-D-galactose is epimerized to α[?]-D-galactose through the action of a mutarotase (Beebe and Freyet al.and Frey, 1998; Thoden and Holdenet al.and Holden, 2002a). α[?]-D-galactose is then metabolized by the Leloir pathway (Leloir 1951), an evolutionarily conserved biochemical pathway which begins with the phosphorylation of galactose by the enzyme galactokinase (GALK) to form galactose-1 phosphate (gal-1P) (Cardini and Leloiret al.and Leloir, 1953). Gal-1P is subsequently, together with the substrate UDP-glucose, converted by galactose-1-phosphate uridylyltransferase (GALT) to form UDP-galactose and glucose-1 phosphate (glu-1P) (Kalckar, Braganca et al., 1953). The Leloir pathway is completed by reversibly forming UDP-glucose from UDP-galactose
2.2. How are the different types of galactosemia detected and diagnosed?
Newborn screening programs worldwide have greatly facilitated the early detection of Galactosemia (Kaye, Accurso et al., 2006; Levy 2010). The screening tests often involve the detection of elevated level of blood galactose and/or specific GAL enzyme in the dried blood spots on filter paper. Elevated galactose will detect GALK deficiency and GALT deficiency, but it may not detect GALE deficiency. Other states screen for GALT activity, and may therefore diagnose Type I Galactosemia. However, this screen will miss GALK and GALE deficiency. The final diagnosis is secured once the specific enzyme deficiency is confirmed by enzymatic assays or by DNA genotyping; these tests are available commercially in the USA (http://www.ncbi.nlm.nih.gov/sites/ GeneTests/, Tests #3437, #2229 and #53782).
2.3. What are the current treatments for galactosemia, and what is the outlook for patients?
The main aspect of management for all forms of Galactosemia is withdrawal of lactose/galactose from the diet as soon as the diagnosis is made, or even considered (Segal 1995). In infants, this means the replacement of breast/cow milk with soy-based formula. However, it has become clear that, despite early detection and (early) dietary intervention, there still is a significant burden of the disease, particularly for Classic Galactosemia where chronic problems persist through adulthood. The most common medical complications of Type I Galactosemia are speech dyspraxia, ataxia, premature ovarian insufficiency, and intellectual deficits, [ANY OF?](yes) which are rarely seen in other forms of galactosemia (Waggoner, Buist et al., 1990 ; Waisbren, Potter et al., 2011). GALK deficiency (Type II Galactosemia) is managed also with lactose/galactose restriction, though the complications are mainly confined to the eye (cataracts) (Bosch, Bakker et al., 2002). GALE deficiency is treated similarly, though complications of this deficiency may not be preventable with such restriction, as is GALT deficiency (Fridovich-Keil, Bean et al., 1993a).
3. Use of SDM to confirm disease-causing mutations in human GALT, GALK, and GALE genes identified clinically
3.1. The issues
Advances in federal and state newborn screening programs worldwide have resulted in the inclusion of the potentially lethal disorder, Galactosemia, in the list of diseases for which newborns are screened. Very often, once an affected newborn is identified by the biochemical assays, it is helpful to know the genotype [ALLELE] of
Unfortunately many patients with Galactosemia identified to-date have novel (private) nucleotide changes in their
3.2. Research design
Our laboratory and others have largely used similar strategies in confirming the suspected human
3.3. The results
The primary goal for expression analysis of the suspected disease-causing mutations in the
3.3.1. Type I (GALT-deficiency) galactosemia
As mentioned above, more than 200 nucleotide changes in the
Due to its frequency among GALT-deficiency galactosemic patients and its association with a poor clinical outcome, the
3.3.2. Type II (GALK-deficiency) galactosemia
More than 20 mutations associated with GALK deficiency have been reported to date. Through SDM studies, the majority of the mutations have been characterized. By expressing 10 variant GALK enzymes in GALK-less
Similarly, Park and colleagues characterized another four missense mutations and one insertion (
3.3.3. Type III (GALE-deficiency) galactosemia
GALE deficiency exists in a continuum, from generalized to peripheral
4. Use of SDM in the understanding of catalytic mechanisms of the human GAL enzymes
4.1. The issue
Although the Leloir pathway is evolutionarily conserved and is indispensable for productive galactose metabolism, the catalytic mechanisms of the GAL enzymes are largely unknown.
4.2. Research design
Several groups have attempted to combine the techniques of SDM, analytical biochemistry and X-ray crystallography to advance the understanding of the catalytic mechanisms of the different GAL enzymes.
4.3. The results
GALK converts galactose to gal-1P by transferring γ?–phosphate group of ATP to the O1 position of galactose. It belongs to a unique kinase superfamily – the GHMP kinase family, which is named after four characteristic family members: galactokinase (GALK), homoserine kinase (HSK), mevalonate kinase (MVK) and phosphomevalonate kinase (PMVK) (Bork, Sander et al., 1993). This family of proteins was first identified by three highly conserved motifs among the four kinases mentioned above by sequence alignment and analysis. Motifs I and III are located at the N-terminal and C-terminal ends; and motif II, the most conserved, is located in the middle of the protein, with the consensus sequence of GLGSS(G/A/S) (Holden, Thoden et al., 2004).
Interestingly, two different catalytic mechanisms have been proposed for this family. A common catalytic strategy to achieve nucleophilic attack is to use a negative charged residue, such as aspartate or glutamate, to act as a Brønsted base. This catalytic base can then abstract a proton from the hydroxyl group of the substrate converting the weakly nucleophilic hydroxyl group into the more strongly nucleophilic alkoxide ion, which then attacks the electron-deficient phosphorus atom in ATP (Fig. 1A). In such systems, it is common to find positively-charged lysine or arginine residues close to the catalytic site to help stabilize the negative charges on the enzyme and the substrates. Studies on MVK suggest this enzyme follows this mechanism. The crystal structure of MVK reveals an aspartate (residue 204 in the rat enzyme) positioned to act as an active site base. There is also a lysine (residue 13 in rat MVK), which is close to both the putative catalytic aspartate residue and the hydroxyl group of the substrate (Fu, Wang et al., 2002; Yang, Shipman et al., 2002). Replacement of the lysine residue with a methionine by SDM resulted in a reduced, but non-zero, rate (Vmax was reduced approximately 60-fold) (Potter, Wojnar et al., 1997). Similar results were observed when the equivalent lysine (residue 18) was changed to methionine in yeast mevalonate diphosphate decarboxylase (Krepkiy and Miziorko and Miziorkoet al., 2004). These results are consistent with this positively-charged residue playing an assisting, but non-vital, role in catalysis. Crystal structures of GALK put it into this mechanism by revealing there are aspartate and arginine residues in the active center close to the galactose C1 hydroxyl group (Asp186 and Arg37 in the human structure, Asp183 and Arg36 in
In contrast, phosphoryl transfer in HSK has been suggested to occur by direct nucleophilic attack on the γ?-phosphate group of ATP by the δ?-hydroxyl of homoserine (Fig. 1B) (Krishna, Zhou et al., 2001). In this mechanism, the latter is stabilized by the formation of a hydrogen bond to a neighboring asparagine residue (Asn141), which is not conserved in the superfamily. Catalysis is proposed to be assisted through activation of the γ?-phosphate of ATP by the magnesium ion, which is coordinated by a conserved glutamate residue (Glu130) with the deprotonation of the δ?-hydroxyl possibly involving the γ?-phosphate (Krishna, Zhou et al., 2001).
GALT catalyzes the transfer of the uridine monophosphate group (UMP) from uridine diphosphate-glucose (UDP-Glu) to gal-1p to form uridine diphosphate-galactose (UDP-Gal) and glucose-1-phosphate (glu-1P) (Kalckar, Braganca et al., 1953). The reaction follows the double displacement mechanism as shown in Fig. 2 (Arabshahi, Brody et al., 1986). The most characteristic feature of the reaction is forming a covalent UMP-enzyme intermediate (Arabshahi, Brody et al., 1986). The intermediate was isolated by gel permeation chromatography in reaction mixtures containing the enzyme and radiolabeled UDP-Glu, and the radiolabeled intermediate could react with gal-1P or glu-1P to form the corresponding radiolabeled UDP sugar (Wong, Sheu et al., 1977a). This intermediate is very fragile in slightly acidic solutions but quite stable in strong basic solutions (Wong, Sheu et al., 1977a; Yang, and Freyand Frey et al., 1979), which indicates the intermediate is phosphoramides. Further degradation study of this intermediate confirmed that the nucleophile in GALT, to which the uridylyl group is bonded in the uridylyl-enzyme intermediate, is imidazole N3 of a histidine residue (Yang and Frey et al. and Frey, 1979).
Catalytic mechanisms proposed for GHMP kinase.
Substituting each of the 15 histidine residues in
Also, as mentioned earlier, by mutating Gln188 of human GALT (equivalent to Gln168 in
GALE catalyzes the inter-conversion of UDP-Glu and UDP-Gal to finish the Leloir pathway of galactose metabolism. There are four key steps for the reaction of GALE as shown in Fig. 3: (1) abstraction of the 4‘-hydroxyl hydrogen of the sugar by an enzymatic base, (2) transfer of a hydride from C4 of the sugar to the C4 of NAD+ leading to a 4‘-ketopyranose intermediate and NADH, (3) rotation of the resulting 4‘-ketopyranose intermediate in the active site, and (4) return of the hydride from NADH to the opposite face of the sugar (Maitra and Ankeland Ankel et al., 1971). When purified, this enzyme contains tightly bound NAD+, which functions as an essential coenzyme to catalyze the reaction (Darrow and and RodstormRodstrom et al., 1968). The binding of the UDP group is strong, while binding with the galactosyl, glucosyl and 4-ketohexopyranosyl moieties is weak (Kang, Nolan et al., 1975; Wong and Frey and Freyet al., 1977b). Early study on the catalytic mechanism of GALE focused on Lys153, since it is close to the NAD+, and the positively-charged ammonium group of Lys153 may perturb the electron distribution in the nicotinamide ring of NAD+ through charge repulsion upon substrate binding (Swanson and Freyand Frey et al., 1993). Replacing this residue with alanine or methionine renders the inability of the mutant proteins to be reduced by the sugar in the presence or absence of UMP. As a result, the catalytic activities of the mutants decreased by a factor over 1000. Also the purified mutant contained much less NADH as compared with wild type (Swanson and Frey and Freyet al., 1993). These results indicate that Lys153 plays an important role in the UMP-dependent reduction of GALE-NAD+. Further studies identified two more important residues, Tyr149 and Ser124, which are involved in glucose moiety binding (Thoden, Frey et al., 1996). SDM studies on the latter two residues revealed that that Tyr149 provides the driving force for general acid-base catalysis, while Ser124 plays an important role in mediating proton transfer (Liu, Thoden et al., 1997). The crystal structure of human GALE confirmed that Tyr149 (Tyr157 for human GALE) sits at the proper position to interact directly with the 4’-hydroxyl group of the sugar and attracts the proton from the hydoxy group and transfers it to NAD+ (Thoden, Wohlers et al., 2000).
Unlike what was observed for the
5. Use of SDM in the development of novel treatment of Type I (classic or GALT-deficiency) galactosemia
5.1. The issues
Unlike Type II or the peripheral Type III Galactosemia, patients with Type I (GALT-deficiency) Galactosemia, also the most common type of Galactosemia, suffer a range of debilitating long-term complications, which include premature ovarian insufficiency, learning deficits, ataxia and speech dyspraxia (Lai, Elsas et al., 2009; Berry and Elsas and Elsaset al., 2011). The current galactose-restricted diet fails to prevent these complications, and the medical/ patient communities are yearning for a more effective therapy. The causes of these organ-specific complications remain unknown, but there is a strong association with the intracellular accumulation of gal-1P. But what is the source of gal-1P in these patients with Classic Galactosemia if they limit their galactose intake? Recent studies have shown that the patients on a galactose-restricted diet are never really “galactose-free. A significant amount of galactose is found in non-dairy foodstuffs, such as vegetables and fruits (Berry, Palmieri et al., 1993; Acosta and Gross and Grosset al., 1995). More importantly, galactose is produced endogenously from the natural turnover of glycolipids and glycoproteins (Berry, Nissim et al., 1995). Using isotopic labeling, Berry and coworkers demonstrated that a 50kg adult male could produce up to 2 grams of galactose per day (Berry, Nissim et al., 1995; Berry, Moate et al., 2004), 2004). Once galactose is formed intracellularly, it is converted to gal-1P by GALK and in GALT-deficient patient cells. As a result, gal-1P is concentrated more than one order of magnitude above normal, even with strict adherence to a galactose-restricted diet. Accumulation of gal-1P is regarded as a major, if not sole, factor for the chronic complications seen in patients with Classic Galactosemia, as suggested by both clinical observation and experimental results from yeast models. Patients with inherited deficiency of GALK, who do not accumulate gal-1P, do not experience the brain and ovary complications seen in GALT-deficient patients (Gitzelmann, Wells et al., 1974; Gitzelmann 1975; Stambolian, Scarpino-Myers et al., 1986). While
5.2. Research design
For the past few years, our group has conducted high-throughput screening (HTS) of small molecule compounds, which could inhibit human GALK enzyme
5.3. The results
Selectivity is always one of the most important properties for developing therapeutic kinase inhibitors because of potential side-effects from unwanted inhibition of other kinases. During the characterization phase of our hit compounds, we found six compounds that selectively inhibit GALK but not any of the other GHMP kinases. These included MVK, which shares a high degree of structural similarity with GALK (Tang, Wierenga et al., 2010). In order to understand what structural elements conferred the specificity of these compounds, we aligned the crystal structure of human GALK and human MVK and focused on the ATP-binding site. Eight amino acid residues and the L1 loop were found to be different in these two kinases. SDM was employed to mutate each residue individually or the L1 loop, and the effects of the changes on the inhibitory capabilities of the compounds were tested. Two compounds were found to be affected by the mutation
Our use of SDM in the characterization of promising GALK inhibitors not only helped identify and confirm the amino acids of GALK with which these small molecules interact, but also exemplified a more rapid and cost-effective way to study the structural interactions between small molecule modifiers and their targets. This novel approach is particularly useful when large-scale co-crystallization projects are not feasible. These studies paved the way for more in-depth investigations to identify the structural determinants required for the inhibitor selectivity of GALK and GHMP kinases.
6. Concluding remarks
Using the disease Galactosemia as an example, we showed that site-directed mutagenesis (
of ATP (µM)
of Galactose (µM)
|Effects on IC50 of compound 1||Effects on IC50 of compound 4||Effects on IC50 of compound 24|
|W106A||No protein expression||-||-||-||-||-|
|W106T||No protein expression||-||-||-||-||-|
|GALK Loop to MVK Loop||0.1||695.4||1857.3||None||None||None|
|S140G||2.1||8.2||141.9||None||Increased 10-fold||Increased 20-fold|
We acknowledge that we could not have completed this manuscript without the outstanding contributions made by our scientific and clinical colleagues, as well as patient volunteers. Research grant support to Kent Lai includes NIH grants 5R01 HD054744-04 and 3R01 HD054744-04S1.