Blood, 1 January 2003, Vol. 101, No. 1, pp. 345-347
RED CELLS
Brief report
HK Utrecht: missense mutation in the active site of human
hexokinase associated with hexokinase deficiency and severe
nonspherocytic hemolytic anemia
Richard van Wijk,
Gert Rijksen,
Eric G. Huizinga,
Hendrik K. Nieuwenhuis, and
Wouter W. van
Solinge
From the Department of Clinical Chemistry and the
Department of Hematology, University Medical Center Utrecht; Department
of Crystal & Structural Chemistry, Bijvoet Center for Biomolecular
Research, Utrecht University, Utrecht, The
Netherlands.
 |
Abstract |
Hexokinase deficiency is a rare autosomal recessive disease with a
clinical phenotype of severe hemolysis. We report a novel homozygous missense mutation in exon 15 (c.2039C>G, HK
[hexokinase] Utrecht) of HK1, the gene that
encodes red blood cell-specific hexokinase-R, in a patient previously
diagnosed with hexokinase deficiency. The Thr680Ser substitution
predicted by this mutation affects a highly conserved residue in the
enzyme's active site that interacts with phosphate moieties of
adenosine diphosphate, adenosine triphosphate (ATP), and inhibitor
glucose-6-phosphate. We correlated the molecular data
to the severe clinical phenotype of the patient by means of altered
enzymatic properties of partially purified hexokinase from the patient,
notably with respect to Mg2+-ATP binding. These kinetic
properties contradict those obtained from a recombinant mutant brain
hexokinase-I with the same Thr680Ser substitution. This contradiction
thereby stresses the valuable contribution of studying patients with
hexokinase deficiency to achieve a better understanding of
hexokinase's key role in glycolysis.
(Blood. 2003;101:345-347)
© 2003 by The American Society of Hematology.
| |
Introduction |
Hexokinase (HK) catalyses the phosphorylation of
glucose to glucose-6-phosphate (G6P) using adenosine triphosphate (ATP)
as a phosphoryl donor. HK-I is the predominant HK isozyme in tissues that depend strongly on glucose use for their physiological
functioning, such as brain, muscle, and erythrocytes. HK-I
displays unique regulatory properties in its sensitivity to inhibition
by physiological levels of the product G6P and, moreover, relief of
this inhibition by inorganic phosphate
(Pi).1 The recent determination of
structures for human2 and rat3 HK-I has
provided substantial insight into ligand binding sites and subsequent
modes of interaction.2-7
Erythrocytes contain a specific subtype of HK-I (HKr)8
that is encoded by the HK-I gene.9
Erythroid-specific transcriptional control results in a unique red
blood cell-specific cDNA that differs from HK-I cDNA at the
5'-end.10-12 Hexokinase deficiency is a rare disease with
nonspherocytic hemolytic anemia as the predominant clinical feature.
Seventeen families with hexokinase deficiency have been described to
date,13 and only one patient has been characterized at the
cDNA level.14 We now report on the molecular defect
underlying hexokinase deficiency in a previously reported Dutch
family.15
| |
Study design |
Patient Z62 was born from a consanguineous marriage,
that is, from first cousins, and was originally reported in
1983.15 She presented with neonatal jaundice and
transfusion-dependent hemolysis. Residual HK activity in the patient's
erythrocytes, platelets, and lymphocytes was about 25% of
normal,15 in agreement with a generalized HK deficiency
caused by a mutation in HK1. DNA was isolated from
peripheral white blood cells according to standardized procedures. The
erythroid-specific promoter, red blood cell-specific exon 1, and exons
2 to 18 of HK1 of the patient and a healthy control
subject were amplified by polymerase chain reaction (PCR)
(primer sequences available on request). cDNA nucleotide and amino
acid numbering starts at the HK-I start codon, and sequence variations are described according to the mutation nomenclature system.16 DNA sequence analysis was performed with the
appropriate primers as described.17
| |
Results and discussion |
DNA sequence analysis of HK1 of the patient and a
control subject revealed several base changes compared with the
reference sequences (GenBank AF016349-016365) (Figure
1A). Base variations for which both the
patient and control subject were homozygous could represent either
sequence discrepancies or polymorphisms. The c.78G>C and IVS15+27C>T
are postulated dimorphic changes for Leu25 and an intronic
polymorphism, respectively. The c.2039C>G missense mutation in exon
15, however, was a likely candidate to cause hexokinase deficiency
because the consequent T680S substitution it encodes affects a critical
residue in the active site2,3,7,18 that is, moreover,
evolutionary conserved (Figure
2A). Subsequent screening of the
patient's family for c.2039C>G affirmed cosegregation of this
mutation with lowered HK activity (Figure 1B), whereas in a healthy
control population (n = 50), this mutation was not encountered (not
shown). We designated the variant HK found in this family HK
Utrecht.
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| Figure 1.
Novel homozygous c.2039C>G (Thr680Ser) missense
mutation in HK1 is associated with decreased HK
activity.
(A) DNA sequence variations in HK1 in both the patient and a
healthy control revealed 5 base changes that represent either
polymorphic substitutions or sequence discrepancies and, in addition, 2 postulated polymorphisms (c.78G>C and IVS15+27C>T). A homozygous
c.2039C>G mutation is detected only in the patient, and the consequent
Thr680Ser substitution affects a highly conserved and structurally
important residue in the active site of HK-I. (B) In the family
pedigree chart, individuals heterozygous or homozygous for the
c.2039C>G mutation are indicated by half-filled and filled symbols,
respectively. Lanes in the agarose gel for each family member are
directly below the symbol for that individual. The agarose gel below
shows a 301-bp PCR product encompassing exon 15, which was amplified
from genomic DNA and subjected to AciI
digestion. c.2039C>G creates an additional restriction site
upon the 2 normally present in this fragment. Thus, digestion of the
wild-type allele results in fragments of 14, 221, and 66 bp, whereas
the extra AciI recognition sequence yields additional
fragments of 34 and 187 bp. Digestion fragments are indicated
by arrows on the right (14 and 34 bp, fragments not shown).
Homozygosity for c.2039C>G is confirmed in patient Z62. The patient's
father (Z32), mother (H37), sisters (Z53, Z56, and Z66.)15
and daughter N84 were all heterozygous, whereas sister Z56 didn't
carry the mutation. All family members heterozygous for the
Thr680Ser substitution displayed reduced HK activity in their
red blood cells, ranging from 0.64-0.89 U/gHb (reference value:
1.34 ± 0.42 U/gHb).15 HK activity for sister Z56. was
1.22 U/gHb. *indicates Z62; M, marker; NC, healthy control.
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| Figure 2.
Thr680 is a critical residue in the active site of HK and is highly
conserved among hexokinases from other species.
(A) Amino acid alignment of the region surrounding human HK-I residue
Thr680 among 30 hexokinases. Where applicable, specific isozymes are
denoted. Conserved residues compared with human HK-I are in shaded
gray. The "c" in parentheses refers to the C-terminal domain of HK.
(B) Schematic drawing of the active site of HK-I shows Thr680, bound
glucose, and adenosine diphosphate (ADP) in ball-and-stick
representation (atom coloring: carbon, black; oxygen, red; nitrogen,
blue; and phosphor, magenta). The carbon atom that is absent in the
Thr680Ser mutant is colored gray. Illustrated is the central position
occupied by Thr680 in the active site where it is located at the tip of
a loop and makes a hydrogen bond with the  -phosphoryl group of ADP.
This figure was generated from the atomic coordinates of the HK-I
ADP/glucose complex7 (protein data bank entry 1DGK) using
computer programs Molscript22 and
Raster3D.23
|
|
Structural changes due to a conservative threonine-to-serine
substitution are likely to be limited since serine can form the same
hydrogen bonds as threonine. However, the consequent removal of the
methyl group could affect hydrogen bond formation by increasing the
rotational freedom of the side chain or, alternatively, by introducing
a water molecule at the site previously occupied by the methyl group.
Small conformational changes could thus have a significant effect on
catalysis, because enzymes typically require a delicate balance of
interactions for optimal activity.
In view of its central position in the active site (Figure 2B),
Thr680 has been proposed to hydrogen bond to the 
- and
-phosphoryl group of ATP during catalysis.7 Thus,
altered binding of phosphate-containing ligands is likely an important
factor in deficient enzyme function. Indeed, enzymatic properties of
partially purified mutant red cell HK from the patient showed near
normal affinity for glucose15 but a 2-fold decrease in
affinity for Mg2+-ATP and a markedly (3- to 9-fold)
decreased affinity for inhibitor glucose-1,6,-diphosphate.15 Surprisingly, these results
contradict with kinetic data obtained from a recombinant human brain
HK-I Thr680Ser mutant as expressed in Escherichia
coli.18 The kcat value of this
mutant decreased only 2.5-fold and was, in fact, characterized solely
by a slightly higher affinity for glucose, whereas the
Km(ATP), Ki(ATP), and
Ki for the G6P analog 1-5-anhydroglucitol 6-phosphate were similar when compared to wild-type
HK-I.18 These unexpected discrepancies must be attributed
either to the different sources of the enzyme, that is, E
coli versus human red blood cells, or to the different N-terminal
ends of HKr and HK-I. It highlights the differences in fate and
function of mutant enzyme in vivo as underscored by a recent case of
glucose-6-phosphate dehydrogenase deficiency.19
The 2-fold higher Km for
Mg2+-ATP15 reflects a relatively modest effect
of the Thr680Ser substitution on ATP binding to the active site. This
change in Km may not affect in vitro HK activity, which is
measured at excess Mg2+-ATP (5 mM),20 but may
have a significant effect in vivo because intracellular ATP
concentrations are much lower (0.6 mM)21 and similar to
the Km. In the study conducted by Ouwerkerk et
al21, blood from patient Z62 was used to measure
intracellular Mg2+-ATP concentrations in oxygenated and
deoxygenated red blood cells. In oxygenated red blood cells,
Mg2+-ATP concentrations were slightly higher in the patient
(1.04 mM) when compared with the healthy control (0.61 mM) and equal to
the reticulocyte control (1.11 mM).21 Likewise, in
deoxygenated red blood cells, comparable Mg2+-ATP
concentrations were measured in the patient (0.57 mM) and healthy
control (0.62 mM),21 which, in fact, denotes a strongly decreased Mg2+-ATP concentration when compared to a
reticulocyte control (1.79 mM)21 and considering the high
degree of reticulocytosis (40%-50%) in the patient. Therefore, we
postulate that at physiological concentrations of Mg2+-ATP,
the 2-fold increase in Km for Mg2+-ATP results
in a significantly decreased HK activity in the patient's red blood
cells, which is reinforced by deoxygenation. These considerations are
in agreement with the severe clinical phenotype as observed in patient
Z62. and even true if HK is expressed and maintained at a normal level.
The residual (in vitro) activity of 25%, however, suggests an
additional effect on either protein expression or enzyme stability.
The identification of a homozygous c.2039C>G (Thr680Ser) missense
mutation in patient Z62 places previously determined enzymatic properties of the mutant enzyme into perspective and stresses the
valuable contribution of studying patients with HK deficiency toward a
better understanding of the key role played by HK in the glycolytic
pathway and, in particular, in red cell metabolism.
| |
Footnotes |
Submitted June 24, 2002; accepted July 29, 2002.
Prepublished online as
Blood First Edition Paper, August 8, 2002; DOI
10.1182/blood-2002-06-1851.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Wouter W. van Solinge, University Medical
Center Utrecht, Department of Clinical Chemistry, G03.550, PO Box
85500, 3508 GA, Utrecht, The Netherlands; e-mail:
wsolinge{at}lab.azu.nl.
| |
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Top
Abstract
Introduction
