Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA
e-mail: wilsonj{at}msu.edu
Accepted 15 January 2003
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Summary |
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Key words: hexokinase, isozyme, subcellular localization, mitochondria, mammalian
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Introduction |
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The initial step in metabolism of Glc through most common pathways is phosphorylation to form glucose-6-phosphate (Glc-6-P) (Fig. 1), the reaction catalyzed by hexokinase. It is evident that indiscriminate metabolism of Glc-6-P through the various potential pathways would not be in the cell's best interest. So what regulates the formation of Glc-6-P and its direction to the metabolic fate that is appropriate for the physiological status of the cell at the time? Are there any differences in the way in which mammalian cells generate Glc-6-P intended for glycolytic metabolism compared with Glc-6-P destined for metabolism via the pentose phosphate pathway? How are cells adapted to favor metabolism of Glc via particular pathways that are related to the role that cell plays in the overall metabolism of the organism? While the answers to such questions are far from clear, selective expression of isozymic forms of HK is likely to be an important factor in determining the pattern of Glc metabolism in mammalian cells/tissues.
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Isozymes of mammalian hexokinase |
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The Types IIII isozymes are 100 kDa molecules thought to have
evolved by duplication and fusion of a gene encoding an ancestral 50 kDa
hexokinase. Thus, these isozymes display internal sequence repetition, and the
N- and C-terminal halves have extensive sequence similarity, both to each
other and to other members of the hexokinase family, which includes the 50 kDa
mammalian Type IV isozyme and 50 kDa hexokinases found in other organisms
(Bork et al., 1993;
Wilson, 1995
;
Cárdenas et al.,
1998
).
Susceptibility to relatively potent inhibition by the product, Glc-6-P, is
generally considered to be an important regulatory feature of the mammalian
Type IIII isozymes, and is also found with 50 kDa hexokinases from
lower organisms such as starfish and the parasite Schistosoma mansoni
(White and Wilson, 1989;
Tielens et al., 1994
). It is
thus likely that sensitivity to inhibition by Glc-6-P evolved prior to the
gene duplication and fusion event that gave rise to the 100 kDa mammalian
isozymes. Duplication and fusion of a gene encoding a 50 kDa Glc-6-P-sensitive
hexokinase should give rise to a gene encoding a 100 kDa hexokinase with both
N- and C-terminal halves having catalytic activity susceptible to product
inhibition. The Type II isozyme has such characteristics
(Ardehali et al., 1996
;
Tsai and Wilson, 1996
), and on
that basis has been suggested to be most closely related to the ancestral 100
kDa hexokinase produced by the gene duplication and fusion event. In contrast,
it is clear that catalytic function resides solely in the C-terminal half of
the Type I and Type III isozymes (White
and Wilson, 1989
; Baijal and
Wilson, 1992
; Tsai and Wilson,
1995
,
1997
). Thus, the Type I and
Type III isozymes likely resulted from further duplication of the gene
encoding an ancestral 100 kDa hexokinase, with subsequent mutations leading to
functional differentiation of the N-terminal halves to serve noncatalytic
(regulatory) functions.
If the capability for phosphorylation of Glc were the sole raison
d'être for hexokinase, then it would seem that a single hexokinase
would be sufficient. Yet, it is intriguing that isozymes of hexokinase exist
even in `simpler' organisms such as yeast
(Rodríguez et al.,
2001). Surely there is more to this than simply making Glc-6-P!
Thus, it is reasonable to conclude that the Types IIV isozymes of
hexokinase play distinct roles in Glc metabolism in mammalian tissues. In the
case of the Type IV isozyme, that role has been rather well defined
(Postic et al., 2001
). What
about the Type I, Type II and Type III isozymes?
As noted previously (Wilson,
1997), one could imagine at least three good (and not mutually
exclusive) reasons for the existence of isozymes: (1) the isozymes may differ
in their catalytic and/or regulatory properties, suiting them for particular
metabolic roles; (2) differences in transcriptional regulation of the isozymes
may permit their selective expression in particular tissues, with distinct
responses to altered metabolic status, e.g. hormonal effects or chronic
changes in physiological activity (Hofmann
and Pette, 1994
); (3) differences in subcellular location of the
isozymes may result in compartmentation of Glc metabolism, with `channeling'
of Glc-6-P to particular metabolic pathways
(Ureta, 1978
;
Ovádi and Srere,
2000
).
Kinetic and regulatory properties of the Types IIII isozymes are
compared in Table 1. The usual
caveats apply to extrapolation of kinetic parameters determined in
vitro to conditions in situ and, as will become evident later,
this may be particularly the case with the Km of the Type
I (and possibly the Type II) isozyme for ATP. Radojkovíc and Ureta
(1987) previously noted that
the Type III isozyme is distinguished by having the highest apparent affinity
for Glc but also the lowest apparent affinity for the other substrate,
ATP as well as being least sensitive to inhibition by the product,
Glc-6-P. The Type III isozyme is also unique in showing substrate inhibition
at higher Glc levels (>approximately 1 mmol l-1), with this
inhibition being antagonized by ATP. Based on the results of Radojkovíc
and Ureta (1987
), and assuming
intracellular [ATP] in the 13 mmol l-1 range, one might
anticipate that any inhibitory effects would be relatively modest at the
concentrations of Glc likely to exist in most cells. Hence, the physiological
significance of these unique regulatory features of the Type III isozyme
remains unclear.
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In contrast, we believe that the distinct response of these isozymes to
Pi provides a useful clue to their physiological roles (Wilson,
1985,
1995
). Specifically, only the
Type I isozyme shows inhibition by Glc-6-P that is antagonized by
Pi. With the Type II and Type III isozymes, Pi is itself
inhibitory and does not antagonize but rather, adds to any inhibition by
Glc-6-P (Wilson, 1995
, and
references therein; Tsai and Wilson,
1995
,
1996
,
1997
). Increase in cellular
[Pi] (due to increased hydrolysis of high energy phosphate
compounds) and decrease in cellular [Glc-6-P] (due to increased flux through
an activated phosphofructokinase reaction) are typically seen during periods
of increased energy demand and associated increase in glycolytic metabolism,
e.g. in brain (Lowry et al.,
1964
). The resulting increase in the [Pi]/[Glc-6-P]
ratio would increase Type I hexokinase activity, leading to the suggestion
(Wilson, 1985
,
1995
) that the Type I isozyme
functions primarily in a catabolic role, introducing Glc into glycolytic
metabolism with the primary purpose of generating energy (ATP). The ubiquitous
expression of the Type I isozyme is consistent with this view, given the
importance of glycolysis in virtually all mammalian tissues. Moreover, the
Type I isozyme is expressed at particularly high levels in brain, a tissue
well known for its virtually total reliance on glycolytic metabolism of Glc to
sustain a high rate of energy metabolism
(Clarke and Sokoloff, 1998
). In
contrast, the Type II isozyme is much more limited in its expression,
primarily being found in insulin-sensitive tissues such as skeletal muscle and
adipose tissue. We have previously argued (Wilson,
1985
,
1995
) that the response of the
Type II isozyme to these same ligands, Glc-6-P and Pi, would better
suit it for an anabolic role, e.g. providing Glc-6-P for resynthesis of
glycogen during recovery of skeletal muscle after contraction. More recent
work is also consistent with an anabolic role for the Type II isozyme as a
source of Glc-6-P for metabolism via the pentose phosphate pathway,
providing NADPH required for lipid synthesis in liver
(Sebastian et al., 2000
) or
lactating mammary gland (Kaselonis et al.,
1999
). By this same reasoning, the similarity in response of the
Type II and Type III isozymes to Glc-6-P and Pi
(Table 1) suggests that the
latter isozyme may also have primarily an anabolic role, but presently there
is no basis for associating the Type III isozyme with a particular anabolic
pathway (or pathways).
Promoter regions governing the transcription of the Type I
(White et al., 1996;
Liu and Wilson, 1997
), Type II
(Mathupala et al., 1995
;
Osawa et al., 1996
;
Heikkinen et al., 2000
) and
Type III (Sebastian et al.,
1999
,
2001
) isozymes have been
isolated and characterized to some extent. It is clear that these are distinct
in character, and responsive to quite different transcription factors. Thus
the existence of multiple genes encoding the isozymes of hexokinase does offer
the flexibility in expression, in different tissues or in different
physiological states, perceived as a benefit of isozymic forms
(Wilson, 1997
).
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Subcellular location and hexokinase function |
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The classic studies of Johnson
(1960) and of Rose and Warms
(1967
) were soon followed by
reports of mitochondrially bound hexokinase from other normal tissues, in
addition to brain, as well as from various tumor cells (for references, see
Wilson, 1985
,
1995
). The potential
physiological significance of this association of a `glycolytic' enzyme with
mitochondria, the primary site for oxidative metabolism, has been the subject
of much speculation. From early on, and particularly after recognition that
the `hexokinase binding protein' of the outer mitochondrial membrane was
identical to porin, and thus that hexokinase might be positioned near the
point at which ATP would exit the mitochondria (and ADP re-enter the
mitochondria), speculation was largely focused on the possibility that this
proximity might foster intimate metabolic interaction between
intramitochondrial oxidative phosphorylation as a source of substrate ATP and
Glc phosphorylation by the mitochondrially bound hexokinase. This had, in
fact, been considered by Rose and Warms
(1967
), but could not be
supported by their experimental results. However, subsequent work by others
(again, for references see Wilson,
1985
,
1995
), using mitochondrially
bound hexokinase from various sources, produced evidence in support of the
view that mitochondrially bound hexokinase had `preferential' or `privileged'
access to intramitochondrially generated ATP.
Work in our laboratory has provided a firm basis for the view that
hexokinase bound to actively phosphorylating brain mitochondria is indeed
tightly coupled to an intramitochondrial compartment of substrate ATP,
generated by oxidative phosphorylation. The experimental support for this
conclusion has been described in a series of publications (BeltrandelRio and
Wilson, 1991,
1992a
,b
;
de Cerqueira Cesar and Wilson,
1995
,
1998
,
2002
;
Hashimoto and Wilson, 2000
). A
complete review of this work is not possible within the constraints of the
present context, but we will highlight some of the principal findings to
illustrate the variety of experimental approaches, all leading to the same
conclusion, that have been utilized in these studies.
Initial studies (BeltrandelRio and Wilson,
1991,
1992a
,b
)
were done using spectrophotometric methods in which ATP production by various
intramitochondrial processes (oxidative phosphorylation, adenylate kinase
reaction, creatine kinase reaction) and Glc phosphorylation by hexokinase were
linked to NADPH production, monitored at 340 nm, by the use of appropriate
coupling enzymes. The basic idea was that by comparing the rate of Glc
phosphorylation with the rate of ATP production from various sources, one
might deduce the relative importance of various intramitochondrial
ATP-generating processes as a source of substrate ATP for hexokinase. And the
conclusion was that, when oxidative phosphorylation was occurring, neither
adenylate kinase nor creatine kinase was a significant source of substrate
ATP. Moreover, only a fraction of the ATP produced by oxidative
phosphorylation was utilized by hexokinase, with the result that [ATP] in the
extramitochondrial medium continued to increase as oxidative phosphorylation
continued. The rate of Glc phosphorylation did not steadily increase in
response to continued increase in extramitochondrial [ATP], however, despite
the fact that the latter was comparable to the Km for ATP
seen with added extramitochondrial ATP in the absence of oxidative
phosphorylation, i.e. the hexokinase would not have been `saturated' with
extramitochondrial ATP as substrate (Fig.
2). This strongly suggested that it was not extramitochondrial ATP
that was being used as substrate.
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Further support for this was provided by results such as those shown in Fig. 3. Here, Glc phosphorylation was monitored after initiation of oxidative phosphorylation by addition of ADP, with increasing concentrations of extramitochondrial ATP present from the start. As expected from classical MichaelisMenten kinetics, the initial rate of Glc phosphorylation increased with increasing extramitochondrial [ATP]. However, with time, a steady state rate of Glc phosphorylation was attained that was independent of the amount of residual extramitochondrial ATP present. These results were interpreted as indicating that, while the mitochondrially bound hexokinase initially used extramitochondrial ATP, initiation of oxidative phosphorylation led to a switch in substrate preference, with hexokinase becoming dependent on an intramitochondrial compartment of ATP in which [ATP] was determined by the rate of oxidative phosphorylation and independent of the extramitochondrial [ATP].
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Although ATP production began almost immediately after initiation of
oxidative phosphorylation by addition of ADP, there was a marked lag in
initiation of Glc phosphorylation by the mitochondrially bound hexokinase
(Fig. 3D) before attainment of
a steady state rate that persisted for an extended period. This initial lag
period was interpreted as the time required to fill an intramitochondrial
compartment with ATP generated by oxidative phosphorylation and from which the
mitochondrially bound hexokinase drew its substrate ATP. Inhibition of
electron transport by addition of KCN resulted in apparent release of ATP,
presumably from the intramitochondrial compartment, and the properties of this
`compartment' (kinetics of filling, linkage to intramitochondrial ATP
production, etc.) were in satisfying agreement with expectations based on this
interpretation (BeltrandelRio and Wilson,
1991,
1992a
,b
).
Unfortunately, agreement with expectations is not an infallible guide to true
fact, and the `apparent release of ATP' was subsequently found to be an
artifact (Laterveer et al.,
1993
), the source of which is still not understood and which could
not be reproduced in later work (de
Cerqueira Cesar and Wilson, 1998
). Despite this discouraging, and
embarrassing, setback, the basic concept of coupling mitochondrially bound
hexokinase to an intramitochondrial compartment of ATP was supported by other
evidence and has withstood subsequent experimental tests.
A double isotopic labeling method was developed as an alternative to the
spectrophotometric procedures (de
Cerqueira Cesar and Wilson, 1995). 14C-Labeled Glc was
used as a substrate for hexokinase, and 32Pi supplied as
substrate for ATP synthesis by oxidative phosphorylation. The
32P/14C ratio in Glc-6-P thus provided a measure of the
specific activity of substrate ATP used by hexokinase. The
32P/14C ratio of Glc-6-P produced by mitochondrially
bound hexokinase was compared with that produced by yeast hexokinase, which
does not bind to mitochondria and thus necessarily utilizes extramitochondrial
ATP as substrate. The kinetics of labeling of the ATP pools used as substrate
by mitochondrially bound and yeast hexokinases were strikingly different,
again consistent with the view that the mitochondrially bound hexokinase was
not utilizing extramitochondrial ATP as substrate, but rather, drawing on an
intramitochondrial compartment of ATP furnished by oxidative phosphorylation.
For example (Fig. 4), addition
of excess 31Pi, thereby markedly decreasing the specific
activity of the 32P-ATP synthesized by oxidative phosphorylation,
resulted in a rapid decrease in the 32P/14C ratio of
Glc-6-P produced by yeast hexokinase, using extramitochondrial ATP. In
contrast, there was a lag and subsequently somewhat slower decrease in the
32P/14C ratio of Glc-6-P produced by mitochondrially
bound hexokinase. The latter observations again were consistent with the view
that the mitochondrial hexokinase was utilizing an intramitochondrial
compartment of ATP that was not freely equilibrated with extramitochondrial
ATP.
|
Still another experimental approach was based on a comparison of the
mitochondrially bound hexokinase and the non-bound yeast enzyme (de Cerqueira
Cesar and Wilson, 1998,
2002
). The underlying logic is
illustrated in Fig. 5. A fixed
amount of brain mitochondria, with bound hexokinase, is mixed with increasing
amounts of rat liver mitochondria, which contain no bound hexokinase. Both
brain and liver mitochondria are actively phosphorylating and thus there is an
increasing rate of ATP production in the system as the amount of liver
mitochondria is increased. By design, the concentration of extramitochondrial
ATP is kept subsaturating, i.e. ÅKm of hexokinase
with extramitochondrial ATP as substrate (in the absence of oxidative
phosphorylation). If the mitochondrially bound hexokinase is using
extramitochondrial ATP as substrate, a progressive increase in the rate of Glc
phosphorylation is expected as the rate of ATP production is increased by
addition of increasing amounts of liver mitochondria. In fact, this is not
what is observed; rather, the rate of Glc phosphorylation by the
mitochondrially bound hexokinase is not significantly affected by increase in
the rate of ATP production (Fig.
6). In contrast, the expected increase in the rate of Glc
phosphorylation is seen if the mitochondrial hexokinase is replaced by an
equivalent amount of yeast hexokinase. These results are thus again consistent
with the view that mitochondrially bound hexokinase is using
intramitochondrial ATP, intrinsic to the mitochondria with which the
hexokinase is associated but independent of any increases in
extramitochondrial ATP emanating from the hexokinase-free liver
mitochondria.
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Finally, further evidence for the view that mitochondrially bound
hexokinase can discriminate between intra- and extramitochondrial ATP comes
from examining inhibition by the Glc-6-P analog, 1,5-anhydroglucitol-6-P
(1,5-AnG6P). In the absence of oxidative phosphorylation and with
extramitochondrial ATP as substrate, 1,5-AnG6P is a rather potent inhibitor,
competitive versus ATP (Fig.
7) (Hashimoto and Wilson,
2000). In contrast, with ATP supplied by oxidative
phosphorylation, 1,5-AnG6P is much less effective as an inhibitor. Clearly,
ATP provided by oxidative phosphorylation is not equivalent to
extramitochondrial ATP. Similar results have recently been reported using the
mitochondrial hexokinase from bovine brain
(de Cerqueira and Wilson,
2002
).
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In short, the present view is that, in the absence of oxidative
phosphorylation, mitochondrial hexokinase can readily use extramitochondrial
ATP, following classical MichaelisMenten kinetics. During active
oxidative phosphorylation, however, the mitochondrially bound enzyme is
coupled to an intramitochondrial pool of ATP, with the rate of Glc
phosphorylation closely correlated with the rate of oxidative phosphorylation.
It seems difficult to believe that, in normal tissue, mitochondria are ever in
a totally nonphosphorylating state. Changes in the rate? Yes, of course,
depending on fluctuations in energy demand. But truly nonphosphorylating?
Probably only under the most dire and ultimately lethal
circumstances. It thus follows that, under normal conditions, the rate of Glc
phosphorylation is closely coordinated with terminal oxidative stages of Glc
metabolism occurring in the mitochondria, with associated production of ATP by
oxidative phosphorylation. As previously noted
(BeltrandelRio and Wilson,
1992a), such coordination may ensure introduction of Glc into
glycolytic metabolism at a rate commensurate with terminal oxidative stages,
avoiding production of neurotoxic lactate
(Marie and Bralet, 1991
) while
ensuring net flux through the cytoplasmic and mitochondrial portions of the
pathway at a rate adequate to meet energy demands
(Fig. 8).
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It is not known how this remarkable change in substrate specificity
(intramitochondrial versus extramitochondrial ATP) is induced by
oxidative phosphorylation, but it is clear that the conformation of the
mitochondrially bound enzyme is affected by mitochondrial membrane potential
as well as other factors related to mitochondrial function, indicating
intimate interaction between the inner (across which the membrane potential
exists) and outer (to which hexokinase is bound) membranes of this organelle
(Hashimoto and Wilson, 2000).
Conformational changes affecting regions of the molecule involved in binding
of substrate ATP had previously been postulated
(de Cerquiera and Wilson,
1998
) to be responsible for changes in substrate specificity.
Type II isozyme
The Type II isozyme also includes a hydrophobic N-terminal sequence capable
of targeting the hexokinase to mitochondria
(Sui and Wilson, 1997). While
association of the enzyme with the `particulate,' presumably mitochondrial,
fraction in homogenates of normal tissues has been reported (for references
see Wilson, 1995
), substantial
amounts are also generally found in the `soluble' fraction and recent
immunolocalization studies (J. E. W., unpublished results) have clearly
indicated the presence of non-mitochondrial Type II hexokinase in certain cell
types in brain. The extent to which mitochondrially bound Type II hexokinase
might be coupled to oxidative phosphorylation has not been, to our knowledge,
examined with mitochondria isolated from normal tissues. This isozyme is also
expressed at high levels in many tumors
(Shinohara et al., 1994
) but,
in view of the aberrant Glc metabolism associated with tumors, it seems
arguable whether the function of Type II hexokinase in tumors can be equated
with that in normal tissues. However, experiments with mitochondrially bound
Type II hexokinase from AS-30D hepatoma cells did indicate `preferred access'
to intramitochondrially generated ATP
(Arora and Pedersen, 1988
).
While the study of Arora and Pedersen
(1988
) demonstrates the
potential for such coupling, extrapolation of these results to the function of
Type II hexokinase in normal tissues may be inappropriate. It would definitely
be of interest to pursue similar studies with Type II hexokinase bound to
mitochondria from normal tissues.
Type III isozyme
The Type III isozyme lacks the hydrophobic N-terminal sequence critical for
targeting to mitochondria. Early reports generally indicated that this isozyme
was found in the `soluble' fraction of tissue homogenates, presumably
indicating a cytoplasmic location, and recent immunolocalization studies have
indicated that Type III hexokinase does exist in cytoplasmic regions of
cerebellar Purkinje neurons (J. E. W., unpublished results). However,
immunolocalization studies have also demonstrated that, at least in many
tissues, the Type III isozyme is associated with the nuclear periphery
(Preller and Wilson, 1992).
The Type III isozyme lacks an obvious classical nuclear targeting sequence,
and the structural feature that determines the perinuclear location has not
been defined. Also undefined are the possible metabolic implications of such a
subcellular location. Given the rather well established metabolic importance
of the binding of Type I (and possibly Type II) hexokinase to mitochondria, it
would seem reasonable to expect that association of Type III hexokinase with
the nucleus may have similar metabolic significance.
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Acknowledgments |
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References |
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