(Received for publication, November 9, 1995; and in revised form, December 1, 1995)
From the
The mammalian hexokinase (HK) family includes three closely
related 100-kDa isoforms (HKI-III) that are thought to have
arisen from a common 50-kDa precursor by gene duplication and tandem
ligation. Previous studies of HKI indicated that a glucose 6-phosphate
(Glu-6-P)-regulated catalytic site resides in the COOH-terminal half of
the molecule and that the NH-terminal half contains only a
Glu-6-P binding site. In contrast, we now show that proteins
representing both halves of human and rat HKII have catalytic activity
and that each is inhibited by Glu-6-P. The intact enzyme and the
NH
- and COOH-terminal halves of the enzyme each increase
glucose utilization when expressed in Xenopus oocytes.
Mutations corresponding to either Asp-209 or Asp-657 in the intact
enzyme completely inactivate the NH
- and COOH-terminal half
enzymes, respectively. Mutation of either of these sites results in a
50% reduction of activity in the 100-kDa enzyme. Mutation of both sites
results in a complete loss of activity. This suggests that each half of
the HKII molecule retains catalytic activity within the 100-kDa
protein. These observations indicate that HKI and HKII are functionally
distinct and have evolved differently.
The four mammalian hexokinases
(ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) initiate
glucose metabolism by catalyzing the conversion of glucose to glucose
6-phosphate (Glu-6-P). ()Hexokinases I-III
(HKI-III) have a molecular mass of 100 kDa, a high affinity for
glucose, and are feedback-inhibited by physiologic concentrations of
Glu-6-P. Glucokinase (HKIV or GK), a 50-kDa protein with a lower
affinity for glucose, is not inhibited by physiologic concentrations of
Glu-6-P, nor is the 50-kDa yeast enzyme. The 100-kDa isoforms are
thought to have arisen from a 50-kDa precursor through gene duplication
and tandem ligation(1) . The high degree of amino acid
similarity between the NH
- and COOH-terminal halves of a
given HK with each other, with yeast HK, and with GK, supports this
hypothesis(2, 3, 4) . Additional support for
a gene duplication event is provided by the demonstration of exon size
conservation between GK and the NH
- and COOH-terminal
halves of HKII and by localization of the likely fusion site within the
HKII gene(5, 6) .
Analysis of the functional
domains of the hexokinases has also been performed to learn how these
enzymes evolved. Glucose and ATP bind to the COOH-terminal half of the
100-kDa HKI molecule, whereas Glu-6-P binds to the
NH-terminal
half(7, 8, 9, 10) . If a yeast-like
HK is the precursor, it is necessary to hypothesize that the catalytic
site is retained in the COOH half, but is lost in the NH
half of HKI and replaced by a regulatory site(1) . The
two halves of the HKI molecule have been obtained by proteolytic
cleavage (11) and by construction of cDNA-based vectors that
direct their expression (12, 13) . As predicted from
studies of the intact molecule, the COOH-terminal half of HKI is
catalytically active, and the NH
-terminal half is
not(11, 12, 13) . However, the observation
that the COOH-terminal half is inhibited by Glu-6-P was not expected
based on the behavior of the intact enzyme(11, 13) .
In view of these observations, a 50-kDa precursor with both catalytic
and regulatory sites, such as exists in various marine organisms,
silkworms, and
schistosomes(14, 15, 16, 17) , could
have been duplicated to form the 100-kDa
hexokinases(11, 13) . If so, the catalytic site in
intact HKI must be lost or occluded in the NH
-terminal half
and retained in the COOH-terminal half, whereas the regulatory site
could involve either or both halves. Although it is assumed that all of
the 100-kDa hexokinases evolved similarly, and that they share
structural and functional features, only the HKI isoform has been
analyzed in detail. We show here that the NH
- and
COOH-terminal halves of rat and human HKII (N-HKII and C-HKII) both
have catalytic activity in the intact enzyme and when expressed as
discrete proteins. In addition, both halves of HKII are inhibited by
Glu-6-P. These observations support the hypothesis that a 50-kDa
precursor with both catalytic and regulatory sites was duplicated to
form the 100-kDa hexokinases, and that HKII retains more of the
characteristics of the ancestral gene than does HKI.
To construct D657A, primers C477 (5`-CATTGTCCAGTGCATCGC-3`)
and C1191 (5`-AGTTCCGACTGTGGCGTTCACCACAGCA-3`) were used in the first
PCR reaction, and primers A2084 (reverse complement of C1191) and B1046
(5`-TCTGAGACAAGAACTTGG-3`) were used in the second PCR reaction. The
third PCR product was digested with XhoI and ClaI and
cloned into pGSTHKII. The PCR fragments inserted into
pGST
HKII were validated by DNA sequencing.
Figure 1:
SDS-polyacrylamide gel electrophoresis
of affinity-purified GST fusion proteins. Each HKII fusion protein was
expressed and purified as described under ``Experimental
Procedures.'' The proteins were stained with Coomassie Blue to
illustrate the purity of a representative preparation of N-HKII, C-HKII
and HKII used to obtain the kinetic data shown in Table 1. The lanes on the left illustrate the relative level of
fusion protein before purification in the crude extracts. The molecular
masses of the protein standards are indicated on the left (in
kDa) and the estimated mass of each purified fusion protein is shown on
the right. The GSTHKII fusion protein has an expected
mass of
130 kDa, GST
N-HKII of
80 kDa, and
GST
C-HKII of
76 kDa.
The purification of the human enzymes
involved the binding of the GST fusion protein to a glutathione
affinity column (Fig. 1). To evaluate whether the GST addition
altered the kinetic properties of any of the enzymes, and to check for
possible species differences, the mRNAs encoding the intact molecule,
as well as the N- and C-terminal halves of rat HKII, were injected into X. laevis oocytes, and HK activity in the cell extracts was
measured. In this cell-based system, as with the purified human HKII
proteins, both the NH- and COOH-terminal halves of rat HKII
actively phosphorylate glucose (Table 2). The kinetic parameters
determined for the rat enzymes are similar to those observed for the
human enzymes ( Table 1and Table 2); thus, the GST addition
does not appear to affect the enzymes. The N-HKII and intact enzymes of
both species have significantly higher affinities for ATP and Glu-6-P
than does C-HKII. In addition, the intact HKII enzymes from both human
and rat have kinetic parameters that resemble those previously observed
in rat skeletal muscle, in which HKII is known to be the predominant
isoform ( (24) and Table 1and Table 2).
No hexokinase activity was observed in extracts obtained after the injection of mRNAs encoding either N-HKII or C-HKII with a single Asp to Ala substitution (corresponding to Asp-209 and Asp-657 in intact HKII) into oocytes (data not shown). These results identify Asp-209 and Asp-657 as critical residues in the catalytic sites of N- and C-HKII, respectively, as is the corresponding amino acid in GK (Asp-205) and the single, COOH-terminal catalytic site in HKI (Asp-657)(13, 25) .
Having identified a single
amino acid mutation that results in the loss of activity of rat N-HKII
and C-HKII, we constructed mutated human HKIIs to evaluate whether the
NH- and COOH-halves are each active in the context of the
intact 100-kDa enzyme. Individual Asp-209
or Asp-657
Ala
mutations (D209A and D657A proteins, respectively) should result in a
partial loss of activity if both halves of HKII are catalytically
active in the intact enzyme. Moreover, an HKII protein with mutations
in both halves (D209A + D657A) should be inactive. These mutant
HKII proteins were expressed as GST fusion proteins and purified as
described for the wild type enzyme. The double mutation resulted in a
complete loss of catalytic activity (D209A + D657A in Table 3). A single mutation in either the NH
- or
COOH-terminal half of HKII (D209A or D657A, respectively) resulted in
about a 50% reduction as compared to the wild type enzyme (84 or 71 versus 147 units/mg, Table 1and Table 3). The
D209A and D657A mutants have K
values for glucose
comparable to those of HKII, N-HKII, and C-HKII (see Table 1and Table 3). D657A has a K
for ATP similar to
that of HKII and N-HKII ( Table 1and Table 3). The D209A
enzyme has a lower K
for ATP compared with that of
C-HKII, but it is still significantly higher than that of HKII ( Table 1and Table 3). These results suggest that the
NH
- and COOH-terminal halves of HKII are active enzymes in
the intact protein, and that the activity of the whole enzyme may be a
combination of the activities of both halves.
Figure 2:
The HKII proteins expressed in X.
laevis oocytes increase cellular glucose phosphorylation. H
O production from
[2-
H]glucose was measured in 7-10 oocytes
at the indicated glucose concentrations 2 days after the injection of
oocytes with GLUT3 mRNA with or without HKII mRNAs. Glucose utilization
of water-injected oocytes was subtracted, at each glucose
concentration, from utilization measured in oocytes expressing the
various proteins. The results are presented as the mean + S.E. of
six experiments. Shown in the inset are the apparent K
and V
values for
glucose utilization.
The present model for the evolution of hexokinases, based on
studies with HKI, suggests that HKI-III evolved by duplication of
a gene encoding a 50-kDa, Glu-6-P-sensitive hexokinase. As a
consequence of the duplication process, the regulatory site in the COOH
half and the catalytic site in the NH half of the 100-kDa
HKs are lost or masked. The observation that both the NH
-
and COOH-terminal halves of HKII have catalytic and Glu-6-P regulatory
functions necessitates a re-evaluation of this hypothesis, at least as
regards this isoform.
The results presented here suggest that HKII
possesses properties that may allow it to play unique roles in glucose
metabolism under different physiological conditions. According to this
hypothesis, each half of HKII may respond to Glu-6-P levels differently
since each has distinct K values for Glu-6-P. In
addition, the two halves of HKII are likely to show different
activities over a range of intracellular ATP concentrations. Therefore,
although the NH
- and COOH-terminal halves of HKII carry out
the same basic reaction in the intact enzyme, they may respond
differently to substrate and inhibitor concentrations. These intriguing
properties of HKII and their possible physiological significance
warrant further investigation.