From the Division of Medical Genetics, Departments of Biochemistry and Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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The substitution of arginine for
glutamine at amino acid 188 (Q188R) ablates the function of human
galactose-1-phosphate uridyltransferase (GALT) and is the most common
mutation causing galactosemia in the white population. GALT catalyzes
two consecutive reactions. The first reaction binds UDP-glucose
(UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the
UMP-GALT intermediate. In the second reaction, galactose-1-phosphate
(gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free
enzyme is recycled. In this study, we modeled glutamine, asparagine,
and a common mutation arginine at amino acid 188 on the
three-dimensional model of the Escherichia coli GALT-UMP
protein crystal. We found that the amide group of the glutamine side
chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP
with lengths of 2.52 and 2.82 Å. Arginine and asparagine could provide
only one hydrogen bond of 2.52 and 3.02 Å, respectively. To test this
model, we purified recombinant human Gln188-,
Arg188-, and Asn188-GALT and analyzed the first
reaction in the absence of gal-1-P by quantitating glu-1-P released
using enzyme-linked methods. Gln188-GALT displaced 80 ± 7.0 nmol glu-1-P/mg GALT/min in the first reaction. By contrast,
both Arg188- and Asn188-GALT released more
glu-1-P (170 ± 8.0 and 129 ± 28.4 nmol/mg GALT/min,
respectively). The overall, double displacement reaction was
quantitated in the presence of gal-1-P. Gln188-GALT
produced 80,030 ± 5,910 nmol glu-1-P/mg GALT/min, whereas the
mutant Arg188- and Asn188-GALT released only
600 ± 71.2 and 2960 ± 283.6 nmole glu-1-P/mg GALT/min,
respectively. We conclude from these data that glutamine at position
188 stabilizes the UMP-GALT intermediate through hydrogen bonding and
enables the double displacement of both glu-1-P and UDP-Gal. The
substitution of arginine or asparagine at position 188 reduces hydrogen
bonding and destabilizes UMP-GALT. The unstable UMP-GALT allows
single displacement of glu-1-P with release of free GALT but
impairs the subsequent binding of gal-1-P and displacement of
UDP-Gal.
The enzyme galactose-1-phosphate uridyltransferase
(GALT)1 (EC 2.7.7.12)
catalyzes the conversion of UDP-glucose (UDP-Glu) and
galactose-1-phosphate (gal-1-P) to form glucose-1-phosphate (glu-1-P)
and UDP-galactose (UDP-Gal) in the evolutionarily conserved Leloir
pathway of galactose metabolism (1). The enzymology of GALT has been
intensively studied using GALT purified from Escherichia
coli (2, 3). Both E. coli and human GALT enzymes catalyze the conversion of UDP-Glu and gal-1-P to UDP-Gal and glu-1-P
via a double displacement mechanism (2-4) (Fig.
1). Under normal physiological
conditions, UDP-Glu binds to GALT to form a GALT-UDP-Glu intermediate.
Glu-1-P is subsequently released, whereas the GALT enzyme remains bound
to UMP. This GALT-UMP intermediate has been isolated and crystallized
(5-8). Gal-1-P then reacts with the GALT-UMP complex to form UDP-Gal,
freeing the GALT enzyme for continued catalysis. GALT does not use
nucleoside di- or triphosphates as nucleotidyl donor substrates, making
it unique among nucleotidyltransferases that utilize phosphates as
acceptor groups (9). The GALT proteins are evolutionarily conserved;
with an overall amino acid identity between the human and E. coli GALT proteins of 46%. There is near 100% conservation of
the amino acid sequence in the catalytic domain studied here (10,
11).
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Fig. 1.
Double displacement reactions of GALT.
Both E. coli and human GALT enzymes catalyze the conversion
via a double displacement mechanism (2-4). Under normal
physiological conditions, GALT binds to UDP-Glu to form a GALT-UDP-Glu
intermediate. glu-1-P is subsequently released, whereas the enzyme
remains bound to UMP. gal-1-P then reacts with the enzyme-UMP
intermediate to form UDP-Gal, freeing the GALT enzyme for continued
catalysis. kn and k n denote
rate constants of the forward and reverse reactions.
The human GALT gene encodes 379 amino acids, contains 11 exons, and spans 4 kilobase pairs of genomic DNA (10). Exon VI is the most conserved domain, is composed of 19 amino acids, and is involved in these catalytic reactions (10). To date, 131 sequence changes have been identified in GALT genes from patients with galactosemia (12-15). The most common mutation is a substitution of arginine for glutamine at amino acid 188 (Q188R). This mutation is caused by an A to G transition, changing CAG to CGG in codon 188 of the highly conserved exon VI (10, 12). This Q188R mutation has a frequency of 70% among white newborns with galactosemia, and homozygosity for the Q188R mutation has been associated with poor prognosis of the disorder (16). The precise pathobiology of this mutation remains unknown. The Gln188 is highly conserved and adjacent to three other conserved amino acids, histidine-proline-histidine at amino acids 184-186, which provide a binding domain and source for the initial nucleophilic attack in the E. coli GALT (17).
A few human GALT mutations, including the Q188R mutation, have been
characterized by various in vitro expression systems (12, 19-21, 36). These studies described some kinetic parameters (apparent KM or Vmax) of purified
mutant proteins but did not explore the normal and mutant mechanisms
involved in position 188 at the biochemical level. In this study, we
used purified, recombinant human GALT proteins, computer modeling,
biochemical assays, and kinetic analyses to clarify the biochemical and
molecular mechanisms by which glutamine 188 contributes to the overall
reaction and how the Arg188 and Asn188
substitutions alter human GALT function.
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EXPERIMENTAL PROCEDURES |
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Computer Modeling of GALT Mutations-- The computer software SYBYL (Tripos Associates) was used to visualize and model human GALT at the active site of uridylated-GALT crystal of E. coli (6-8). At the beginning of this study, the Protein Data Base file of the E. coli uridylated-GALT crystal, 1HXQ, deposited at the Brookhaven's National Laboratory was "on hold." We were graciously given the Protein Data Base file by the authors, Drs. J. Wedekind, Perry A. Frey, and Ivan Rayment from the Institute for Enzyme Research, University of Wisconsin. All computer modelings were performed at the Biomolecular Core Facility (Department of Biochemistry, Emory University) with consultation from Dr. Kim Gernert.
Components of the Bacterial Expression System for GALT--
The
bacterial host strain E. coli BL21(DE3)
[F ompT hsdSB
(rB
mB
) gal dcm (DE3)] used in
this expression study was purchased from Novagen. The gal
operon of BL21(DE3) was disrupted by transposon mutagenesis to produce
a "GALT-less" background for human GALT expression and purification
in the transfected bacteria.
The human wild type Gln188-GALT cDNA was cloned into
vector pGEM3zf() (Promega) (22) and was amplified by polymerase chain reaction with primers GALTPRO5 and GALTPRO3 (see Table I). The amplified polymerase chain reaction product was digested with restriction enzymes BamHI and HindIII and
subcloned into the bacterial expression vector pTrcHisA (Invitrogen),
which allowed high level of expression of the cloned
Gln188-GALT cDNA in E. coli. Ampicillin
resistance was conferred to the transformed bacteria E. coli
BL21(DE3) strain, which enabled 100% selection. The resultant
recombinant vector was named pKL1. To ensure that no mismatches were
introduced during polymerase chain reaction, we sequenced the cloned
Gln188-GALT cDNA twice in both forward and reverse
orientations using protocols for the ABI Prism 310TM
Genetic Analyzer Sequencing System (Applied Biosystems). To amplify the
entire GALT cDNA, we used primers EXON2FOR, EXON3REV, EXON5FOR, EXON6FOR, EXON6REV, EXON9FOR, and EXON10FR (see Table I).
We introduced the Q188R and Q188N mutations into the wild type
Gln188-GALT cDNA sequence in pKL1 by the polymerase
chain reaction-based site directed mutagenesis protocol developed by
Stratagene (Quick ChangeTM) and the corresponding
mutagenesis primers (see Table I). The change from glutamine to
asparagine required changes in two bases in the codon 188 (CAG AAC). This was first achieved by site-directed mutagenesis of the wild
type Gln188-GALT cDNA cloned in pKL1 to form pACWK
using mutagenesis primers Q188KP1 and Q188KP2 (see Table I). It was
then followed by the site-directed mutagenesis of the mutant GALT
cDNA in pACWK to form pACWN using mutagenesis primers Q188NP1 and
Q188NP2 (Table I).
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After mutagenesis, the parental wild type plasmids were digested with DpnI, leaving only the mutant plasmids. Both the wild type and mutant plasmids were transformed into competent E. coli BL21(DE3) by the CaCl2 method (23). The presence of the Arg188 or Asn188 mutation in the mutant plasmid was confirmed by restriction analyses and direct DNA sequencing of plasmid DNA. Both mutations eliminated the natural BstNI restriction site at codon 188.
Induction of GALT cDNA Expression-- To induce expression of recombinant human GALT in transformed E. coli BL21(DE3), we cultured bacteria in 5 ml of LB broth containing 50 µg/ml ampicillin overnight. The next morning 1 ml of the overnight culture was inoculated into 1 liter of fresh, identical medium. The culture was allowed to grow at 37 °C until A600 = 1.0. Then, isopropyl-D-thiogalactoside (Sigma) was added at a final concentration of 1 mM to induce GALT synthesis, and the culture was incubated for an additional 3 h. Cells were then harvested by centrifugation at 6000 × g for 10 min.
Purification of the Recombinant His6-tagged Human GALT Proteins-- The bacterial expression vector, pTrcHisA cotranslates a hexamer of histidines (His6) in frame at the amino terminus of the cloned GALT cDNA. A nickel-charged resin affinity column was used to purify recombinant GALT from bacterial cell lysates using modification of the manufacturer's method (Qiagen) (24, 25). Lysates of bacterial pellets were incubated with nickel-charged resins at 4 °C for 45 min with gentle shaking. Nonspecific binding of non-His6-tagged proteins were eluted with wash buffer containing 20 mM imidazole. The His6-tagged, Gln188-, Arg188-, or Asn188-GALT proteins were then eluted with 100 mM imidazole. Eluates were concentrated and desalted using Centricon-300 and resuspended in a small volume of glycine buffer (100 mM, pH 8.7). Protein concentrations were determined by Bio-Rad assay with bovine serum albumin standards.
Western Blot Analysis of Purified His6-tagged GALT Proteins-- GALT proteins were identified by a rabbit anti-human GALT polyclonal antibody using a Western protocol described previously (26).
Assay of GALT Enzyme Activity--
Assays for the overall,
double displacement reaction were carried out at 37 °C in 1 ml of
glycine buffer (100 mM, pH 8.7) containing 0.6 mM UDP-Glu, 5 mM MgCl2, 5 mM dithiothreitol, 0.8 mM NADP, 1.2 mM gal-1-P, 5 µM glucose--1,
6-diphosphate, phosphoglucomutase (0.5 IU/ml), glucose-6-phosphate
dehydrogenase (0.5 IU/ml) (27). The formation of NADPH was quantitated
by absorbance change at 340 nm. The quantitative relationship between
increase in NADPH production and glucose-1-phosphate released was
quantitated using the Beer-Lambert equation: Absorbance =Concentration
of solutes × Optical path length × Molar coefficient of
extinction. In this study, the solute was NADPH, the optical path
length was 1 cm, and the Molar coefficient of extinction for NADPH at
340 nm was 6220 M
1 cm
1. We
calculated
Abs340 (change in the absorbance) =
C (change in the concentration) × 1 × 6220.
To quantitate the single displacement reaction solely, gal-1-P was replaced with distilled water. All chemicals and enzymes were purchased from Sigma.
Mathematical Analyses of Rate Constants for the Single
Displacement Reaction--
The microcomputer program, SSREG.BAS
(steady-state rate
equation generator) (28) composed and
generously provided by Dr. Robert Gunn (Physiology Department, Emory
University) was used to analyze the rate constants for the single
displacement reaction. This program is based on the algorithm of Indge
and Childs (29), which is a modification of King and Altman's
graphical method for derivation of rate equations (30).
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RESULTS |
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Computer Modeling of Human Gln188-, Arg188-, and Asn188-GALT-- To develop an hypothesis for the structural role of the conserved amino acid Gln188 in human GALT, we modeled E. coli GALT-UMP coordinates at amino acid Gln168 (Fig. 2). There are 20 more amino acids at the amino terminus of human GALT protein as compared with the E. coli GALT (10, 11). Thus, human Gln188 is equivalent to amino acid Gln168 of E. coli GALT (Fig. 2). We found that the amide group of the glutamine side chain provided two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82Å (Fig. 3A). When arginine was substituted for glutamine at position 188 of the human GALT protein, the model identified only one hydrogen bond of 2.52 Å from the amide side chain of arginine (Fig. 3B). However, one could argue that although arginine substitution disrupted normal formation of the hydrogen bonds with UMP, it has a charge change that might alter the local GALT conformation. To control for the potential effects of this charge change, we substituted asparagine for glutamine at position 188. Asparagine is isofunctional with respect to glutamine, except its side chain is one carbon atom shorter. In addition, when we modeled the asparagine 188 mutation, only one hydrogen bond of 3.02 Å was formed (Fig. 3C). So if glutamine stabilizes and properly orients the GALT-UMP mainly by the two hydrogen bonds, we would expect asparagine substitution to show similar deleterious results as the natural mutation Q188R. Furthermore, if glutamine 188 stabilizes the GALT-UMP intermediate, we would expect that asparagine or arginine substitution for glutamine would interfere with gal-1-P binding and the subsequent release of UDP-Gal in the second displacement reaction but not the release of glucose-1-phosphate in the first reaction. To further investigate these hypotheses, we dissected and analyzed the two displacement reactions in vitro using purified Gln188-, Arg188-, and Asn188-GALT proteins produced in the transformed bacteria. Purified GALT enzymes, instead of whole cell extracts from the bacterial expression system, were necessary because bacterial extracts contained abundant gal-1-P (data not shown). To isolate and examine the first displacement reaction (see Fig. 5A), we must deplete the reaction of gal-1-P. If gal-1-P is present in the reaction, the products of the first displacement reaction will instantaneously react with gal-1-P and complete the second displacement reaction (Fig. 1).
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The Bacterial Expression System for Human GALT-- We assayed bacterial extracts of transformed ampicillin-resistant, GALT-less E. coli BL21(DE3) for induced GALT activity by measuring the overall double displacement reaction. Bacterial extracts containing wild type Gln188-GALT cDNA had an overall reaction rate of 1338.9 ± 132.6 nmol glu-1-P formed/mg cell extracts/min (n = 6) (see Table III). By contrast, when bacteria were transformed with Arg188- or Asn188-GALT cDNA, the overall reaction rate for their extracts was 3.2 ± 1.18 or 18.75 ± 6.6 nmol glu-1-P formed/mg cell extracts/min (n = 6), respectively (see Table III). The difference between the results from the mutant Arg188- and Asn188-GALT were significant, with p = 0.00534. These results were similar to the GALT biochemical phenotypes seen in erythrocytes from normal and galactosemic patients who are homozygous for the Q188R mutation (10, 12). These results also show that the His6 epitope tag fused at the amino terminus of the Gln188-GALT did not alter the catalytic function, which is in agreement with an earlier report that the amino terminus epitope-tagged, wild type Gln188-GALT was fully functional (31).
Purification of the Recombinant Human GALT Proteins-- Next we purified the recombinant human Gln188-, Asn188-, and Arg188-GALT proteins by affinity chromatography (24, 25) (Table II). In Fig. 4A, the three left lanes next to the molecular mass markers contained Gln188-GALT (pKL1) eluted with increasing concentrations of imidazole (100, 150, and 250 mM). Two prominent Coomassie Blue-stained protein bands are seen in each successive lane. The upper band has the molecular mass of the dimeric form of GALT, and the lower band has the molecular mass of its monomer. These bands were not seen in the three right lanes, which were loaded with eluates prepared from bacteria transformed with control plasmid pTrcHisA containing no GALT cDNA inserts (the negative controls).
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To confirm the identity of the purified proteins, we blotted an identical SDS-PAGE gel to a nytran membrane and performed Western blot analysis using an anti-human GALT polyclonal antibody (26) (Fig. 4B). Two protein species that had molecular masses of the two protein bands on SDS-PAGE reacted with our anti-human GALT polyclonal antibody (26). Because the anti-human GALT polyclonal antibody was raised in rabbits injected with freshly prepared GALT protein isolated from human red blood cells, our Western blot results suggest that recombinant human Gln188-GALT protein shares at least two similar epitopes with native human GALT from human erythrocytes (26). In subsequent kinetic analyses, we assume that both dimeric and monomeric forms of GALT are active (32, 33). The Arg188- or Asn188-GALT were purified using the same procedure and gave identical results regarding protein mass by SDS-PAGE with immunoblotting (data not shown).
The Specific Activity of the Purified GALT Proteins-- Purified GALT proteins were assayed for their capacity to catalyze the overall double displacement reactions in the presence of 600 µM UDP-Glu and 1.2 mM gal-1-P. We found that the specific activity of the wild type Gln188-GALT was 80,300 ± 5,910 nmol glu-1-P/mg GALT/min (n = 3) (Table III). By contrast, the specific activities of mutant Arg188- and Asn188-GALT were 600 ± 71.2 (n = 3) and 2960 ± 283.6 (n = 3) nmol glu-1-P/mg GALT/min, respectively (Table III). Apparently arginine substitution had a more deleterious effect than asparagine substitution on bi-bi molecular GALT reaction.
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The First Displacement Reaction--
We devised a strategy to
separate the first displacement reaction from the overall double
displacement reactions through omitting gal-1-P in the reaction. By
depleting the reactions of gal-1-P, we initially predicted that each
molecule of GALT enzyme would only catalyze the release of the gal-1-P
and form the GALT-UMP intermediate. If no gal-1-P was available for the
second displacement reaction, the overall reaction would stall without
recycling the GALT enzyme for another round (Fig.
5A). If this prediction were true, only one molecule of glucose-1-phosphate would be displaced by
each molecule of GALT. Because glucose-1-phosphate was removed from the
reaction by our enzyme linked assay, quantitation of glucose-1-phosphate released and NADPH subsequently produced should be
stoichiometric. We would then expect a Abs340 = 1.0 to result from a change of NADPH concentration = 1.0/6220
M = 161 µM in the reaction mixture, as
dictated by the Beer-Lambert equation. Because this would predict only
1 mol of NADPH produced from each mole of glucose-1-phosphate released
and because each mole of GALT could only displace 1 mol of
glucose-1-phosphate, we would have to add 1.6 × 10
7 × 47,000 (molecular mass of His6-tagged GALT) = 7.56 mg of
GALT enzyme to produce a change in absorbance of 1.0. However, if the GALT-UMP complex could dissociate to free UMP and GALT molecules and
the free GALT enzyme could recycle only the first reaction with another
UDP-Glu molecule, the single displacement of glucose-1-phosphate could
continue (Fig. 5B). It would be possible under this second hypothesis to achieve a
Abs340 = 1.0 with
less than 7.56 mg of GALT enzyme.
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From the results exemplified in Fig. 6,
only microgram quantities of Gln188-GALT were required to
quantitate the single displacement reaction. Each data point in Fig. 6
represents the mean value of three independent experiments where only 8 µg of purified Gln188-GALT protein was used to assay the
first displacement reaction. There was a steady, significant release of
glucose-1-phosphate over 3 h as compared with the "negative
control" (Fig. 6). The negative control was column eluate of the
bacterial extracts prepared from bacteria, which were transformed with
the expression vector without any GALT cDNA insert. According to
the Beer-Lambert equation, a Abs340 of 0.697 at 175 min would equal to a
C = 11.2 × 10
5 M. For
C = 11.2 × 10
5 M; glucose-1-phosphate produced in the
reaction = 11.2 × 10
8 mol = 112 nmol. The
8 µg of purified GALT protein added to the reaction was equivalent to
only 170 pmol of GALT monomeric subunits (8 × 10
6/47,000 = 1.70 × 10
10
mol = 170 pmol). Thus, some of the GALT-UMP intermediate must dissociate to free UMP and GALT and recycle a single displacement reaction releasing glucose-1-phosphate in the absence of gal-1-P. The
rate of release of glucose-1-phosphate in the first displacement reaction was directly proportional to the amount of the wild type and
mutant enzymes up to 10 µg in a time course of 5 h (data not shown). Eluate prepared from E. coli transformed with the
expression vector alone without any GALT cDNA insert (negative
control) had no significant increase in Abs340
after 3 h of incubation in identical reagents, substantiating the
specificity of the reactions (Fig. 6). When we used
14C-labeled UDP-Glu as substrate in the single displacement
reaction and separated the reaction products by DEAE-cellulose
chromatography, we found that detectable quantities of
[14C]glucose-1-phosphate were formed from micrograms of
GALT, supporting our spectrophotometric findings (data not shown).
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We then examined the first displacement reaction using the purified mutant Arg188- and Asn188-GALT enzymes. We found that the Asn188- and Arg188-GALT catalyzed the displacement of glucose-1-phosphate from UDP-Glu. In fact, the mutant GALT enzymes were faster than wild type Gln188-GALT (Fig. 6). Gln188-GALT produced 80 ± 7.0 nmol glu-1-P/mg GALT/min, whereas the Arg188- and Asn188-GALT enzymes displaced 170 ± 8.0 and 129 ± 28.4 nmol glu-1-P/mg GALT/min, respectively (Table III).
The Double Displacement Reactions-- When gal-1-P was added to the wild type Gln188-GALT to complete the overall double displacement reactions, there was a 1,000-fold increase of glucose-1-phosphate released within a minute (Fig. 6 and Table III). The release of glucose-1-phosphate in the overall reaction was 80,030 ± 5,910 nmol glu-1-P/mg GALT/min, whereas only 80 ± 7.0 nmol glu-1-P/mg GALT/min was formed in the absence of gal-1-P (Table III). This suggested that the bi-bi molecular double displacement reaction was 3 orders of magnitude faster than the isolated single displacement reaction.
When gal-1-P was added to the reaction mixture containing the mutant Arg188- or the Asn188-GALT, there was a less significant increase in the overall reaction (Fig. 6), confirming the need for glutamine 188 to stabilize the UMP-GALT intermediate, bind gal-1-P, and accomplish the second displacement reaction rate (Table III).
Mathematical Analysis of Rate Constants for the Single Displacement Reaction-- We analyzed the rate constants of the first single displacement reaction for both Gln188- and Arg188-GALT in the absence of gal-1-P (Fig. 5B). We used a microcomputer program (28) based on the algorithm of Indge and Childs (29) and King and Altman's original graphical method for derivation of rate equations (39). From this analysis, the overall rate of product formation at steady state is as follows.
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
Although slower than the overall reaction, the single displacement reaction was saturable when catalyzed by either Gln188- or Arg188-GALT (Fig. 7). The Vmax for Arg188-GALT was greater than that of Gln188-GALT (294.1 nmol glu-1-P/mg GALT/min versus 149.3 nmole glu-1-P/mg GALT/min). From Equation 3, one sees that one criterion for the Vmax of Arg188-GALT to be greater than that of Gln188-GALT is that the rate constant k5 of the conversion of the Arg188-GALT-UMP intermediate to free Arg188-GALT and free UMP would be greater than the k5 of the conversion of the Gln188-GALT-UMP intermediate to free Gln188-GALT and free UMP. Thus by formal mathematical deduction, it takes longer for the Gln188-GALT-UMP intermediate to produce free GALT than the Arg188-GALT-UMP intermediate, and thus a lower capacity to recycle when the first displacement reaction is isolated.
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DISCUSSION |
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GALT is of considerable interest to fundamental biochemistry and provides a unique insight into a bi-bi molecular, double displacement reaction (2, 3). Earlier site-directed mutagenesis experiments concluded that amino acids His164 and His166 of the E. coli GALT were crucial for initiating the first displacement reaction, but little is known regarding the requirements for the second displacement reaction (17). Meanwhile, a prevalent missense mutation, Q188R, found in the GALT genes of many galactosemic patients suggested an important catalytic role played by the highly conserved amino acid glutamine at position 188, which is equivalent to glutamine 168 in E. coli GALT (10, 16, 18, 19).
Our initial examination of the structural role of the amino acid glutamine 168 (equivalent to glutamine 188 in human GALT) in the crystal structure of the E. coli GALT-UMP (5-8) showed us that Gln188 in the human GALT could stabilize the GALT-UMP intermediate through two hydrogen bonds formed between the amide side chain of Gln188 and the phosphoryl oxygen of the UMP moiety (Fig. 3A). Because only one hydrogen bond could be formed in the cases of arginine or asparagine substitutions (Fig. 3, B and C), we subsequently proposed that the stabilization of the GALT-UMP intermediate is essential for the subsequent release of UDP-Gal in the second displacement reaction. This hypothesis was substantiated when we dissected and analyzed the two displacement reactions in vitro using purified recombinant Gln188-, Arg188-, and Asn188-GALT enzymes.
Our analysis of the first displacement reaction also showed that the
GALT-UMP intermediate formed could undergo hydrolysis to form free GALT
and UMP and recycle UDP-Glu to glucose-1-phosphate (Fig.
5B). Microgram quantities of GALT were capable of catalyzing this first displacement reaction, whereas milligram quantities would
have been required if no recycling occurred. These data supported the
earlier studies of the E. coli GALT defining nucleophilic His166 N2 attack by E. coli GALT on the
GALT-bound UDP-Glu substrate and consequent displacement of
glucose-1-phosphate to form the GALT-UMP intermediate (5-8). A
"covalent" bond was modeled between the UMP moiety and the amino
acid His166 of the E. coli GALT in the
uridyl-GALT intermediate (5-8). These investigators emphasized that
there was a substantial positive charge build-up on the
His166 imidazolium ring (5-8), suggesting that
uncatalyzed hydrolysis of imidazolium-UMP between pH 5 and
pH 10 could occur, and such uncatalyzed hydrolysis was further enhanced
by the positive charge built up in the imidazolium group (37). Because
we excluded gal-1-P, it was no longer available to this charged
imidazolium group. Furthermore, glucose-1-phosphate was removed by the
enzyme-linked reactions. Thus, the covalent bond formed by the
zwitterionic UMP-imidazolium group was subjected to uncatalyzed
hydrolysis resulting in the formation of free GALT and free UMP. To
support this notion, we found that when the mutant
Arg188-GALT was used, a higher Vmax
for the glucose-1-phosphate release in the first displacement reaction
was observed, supporting the thesis that Arg188-GALT forms
a less stable GALT-UMP intermediate than Gln188-GALT, and
therefore faster recycling than with Gln188-GALT was used
(Fig. 7).
Our assay for the first displacement reaction using the purified wild type and mutant GALT also showed that the mechanism of dysfunction caused by the Q188R and Q188N substitutions did not impair binding of UDP-Glu or displacement of glucose-1-phosphate from GALT-UDP-Glu (Fig. 1). These results are in agreement with the crystallization studies of the E. coli GALT (35) and our kinetic analysis of the Arg188-GALT and Gln188-GALT of humans for the first displacement reaction (Fig. 7). In the E. coli GALT-UMP crystal, it was shown that Gln168 was not involved in the initial binding of UDP-Glu (35). Our kinetic studies of the human GALT showed that both purified proteins were saturable with UDP-Glu, and although Arg188-GALT had a greater Vmax than Gln188-GALT, both enzymes had the similar KM of about 400 µM (Fig. 7). Thus by computer modeling and kinetic analyses of the first displacement reaction, reduced hydrogen bonding was a rational explanation for the observed enhanced recycling capacity.
By contrast, when the overall reaction was analyzed through release of
glucose-1-phosphate, the substitution of either arginine or asparagine
for glutamine at amino acid 188 severely impaired GALT capacity (Table
III). It thus appears that to accomplish the overall double
displacement reaction, a stable GALT-UMP intermediate is required to
bind gal-1-P and release UDP-Gal. This phenomenon is better
accomplished by two hydrogen bonds to the phosphoryl moiety of UMP from
the amide side chain of glutamine than by the single hydrogen bonds
available from arginine or asparagine (Fig. 3).
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ACKNOWLEDGEMENTS |
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We are grateful to Sharon D. Langley for technical assistance in DNA-direct sequencing of the plasmids. We thank Profs. Perry A. Frey and Ivan Rayment (Institute for Enzyme Research, University of Wisconsin) for providing the unpublished coordinates of the E. coli GalT crystal. We are also indebted to Drs. Robert Gunn and Kim Gernert (Emory University) for consultations on rate constants analyses and computer modeling.
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FOOTNOTES |
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* This work was supported in part by U. S. Health and Human Services Grant Grant PO-1 HD29847-04 from the National Institute of Child Health and Human Development (to L. J. E.) and by U. S. Public Health Services Grant M01-RR00039 (to L. J. E.) for the General Clinical Research Center of Emory University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Medical
Genetics, 2040 Ridgewood Dr. Bldg., Emory University School of Medicine, Atlanta, GA 30322. Tel.: 404-727-5786; Fax: 404-727-5783; E-mail: lje{at}rw.ped.emory.edu.
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ABBREVIATIONS |
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The abbreviations used are: GALT, galactose-1-phosphate uridyltransferase; UDP-Glu, UDP-glucose; gal-1-P, galactose-1-phosphate; glu-1-P, glucose-1-phosphate; UDP-Gal, UDP-galactose; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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