Where is hexokinase

A hexokinase is an enzyme that phosphorylates hexoses, which are sugars with six carbons, resulting in hexose phosphate. Glucoseis is the most important substrate of hexokinases in most organisms, while glucosephosphate is the most important product. Hexokinase is able to transfer an inorganic phosphate group from ATP to a substrate.

There are genes that encode hexokinase in every domain of life. They can be found in bacteria, plants and vertebrates, including humans. More than one isoform or isozyme can occur in one species, providing different functions.

Hexokinases are actin fold proteins and share a common ATP binding site core surrounded by more variable sequences.

When a hexose is phosphorylated it is often limited to several intracellular metabolic processes. The differences in these two conformations allows glucokinase to function properly in different levels of glucose concentration. Proposed Mechanism for Glucokinase: As described above, glucokinase has a distinct conformation change from the active and inactive form. Experiments have also shown an intermediate open form based on analysis of the movement between the active and inactive form.

The switch in conformations between the active form and the intermediate is a kinetically faster step than the change between the intermediate and the inactive form. The inactive form of gluckokinase is the thermodynamically favored unless there is glucose present.

Glucokinase does not change conformation until the glucose molecule binds. The conformation change may be triggered by the interaction between Asp and the glucose molecule.

Once glucokinase is in the active form, the enzymatic reaction is carried out with the presence of ATP. The experiments suggest that glucokinase is found in hepatocyte nuclei and are found inactive at low plasma glucose levels, but found active when higher glucose levels are present.

GKRP would then would likely be an allosteric inhibitor of glucokinase that specifically binds to the inactive form of glucokinase.

The crystal structures of the glucokinase-GKRP complex are being determined to clearly identify the interactions between glucokinase and glucokinase regulatory protein. Role in Organ Systems: In the liver glucokinase increases the synthesis of glycogen and is the first step in glycolysis, the main producer of ATP in the body.

Glucokinase is responsible for phospohorylating the majority of glucose in the liver and pancreas. Glucokinase only binds to and phosphorylates glucose when levels are higher than normal blood glucose level, allowing it to maintain constant glucose levels [4]. By phosphorylating glucose, glucokinase creates glucose 6-phosphate.

Glucose 6-phosphate can then be used by the liver through the glycolytic pathway. Along with this process in the liver, glucokinase also facilitates glycogen synthesis. Through this the majority of the body's glucose is stored. Glucose 6-phosphate is also one of the starting materials of the TCA cycle which is responsible for the majority of ATP production in the body. In the pancreas, a rise in glucose levels increases the activity of glucokinase causing an increase in glucose 6-phosphate.

This causes the triggering of the beta cells to secret insulin [5]. Since glycogenolysis involves production of GP, which inhibits HKs [21] , [22] , we postulated that elevated GP levels during glycogenolysis might be responsible for inhibiting HK activity, so that intracellular glucose clearance is delayed. Consistent with this hypothesis, Fig. However, after a 75 s exposure to 10 mM glucose Fig.

In contrast, exposure to 1 mM extracellular glucose for several minutes, which increased intracellular glucose level by less than half compared to 10 mM glucose, did not cause a delay in glucose metabolism Fig. Panel A illustrates how exposure to 10 mM extracellular glucose for 75 s 1 , 30 s 2 and 50 s 3 affects intracellular glucose clearance. In the 3 cases CytoB was applied for 15 s prior to removal of extracellular glucose.

The 3 rates are compared in the right hand side panel and show that the final rate of glucose clearance is similar in the 3 cases and that long exposure to extracellular glucose delays the clearance. In B a comparison of data obtained with 10 mM and 1 mM extracellular glucose show that application for up to two minutes of 1 mM glucose had no effect on the rate of intracellular glucose clearance, demonstrating that intracellular glucose must reach a threshold to induce this effect.

The right hand side panel shows again that the final rate of glucose clearance measured in the presence of 10 mM glucose is similar to the maximum rate measure with 1 mM and the effect of high glucose is thus to delay clearance.

To test this hypothesis further, we imaged glycogen stores directly by using probes linking either mCherry or GFP to PTG and G L , both of which are part of the family of glycogen targeting subunits of PP-1 [27].

One day after transfection, cells were incubated for 2 to 3 hours in the absence of glucose to deplete glycogen stores. In the absence of GLUT1 overexpression, some CHO cells exhibited dim homogenous fluorescence, while others had a few small and bright punctuate deposits Fig.

After re-addition of glucose 10 mM for 30—60 min, the number, size and brightness of small glycogen deposits began to increase. This phenomenon intensified over 24 hrs, until the deposits fused and partially filled the cell Fig. After a two hour exposure, removal of glucose resulted in a rapid disappearance of glycogen deposits Fig. With overexpression of GLUT1, the rate of appearance of glycogen dramatically increased, such that small deposits were observed within a few minutes of exposure to 10 mM glucose Fig.

In Panel A and D the cells were incubated in the absence of glucose for 2 to 3 hours prior to beginning cell imaging. This incubation in the absence of glucose was carried out to deplete preformed glycogen. Without GLUT1 overexpression glycogen build up was slow, occurring over several hours.

This build up proceeded for up to 24 h filling almost completely the cell in some cases C. With GLUT1 overexpression the rate of glycogen synthesis increased and glycogen deposits could be observed 5 min after exposure to 10 mM glucose outside. It is interesting to note that glycogen deposition occurred at least initially near the nucleus where mitochondria aggregate, suggesting that GP generated by glycogen degradation may be directly fed on to mitochondria. Thus, PTG, which stimulates glycogen synthesis by facilitating the interaction of the regulatory enzyme PP-1 protein phosphatase-1 with GS, GP and phosphorylase kinase, leads to greater glycogen synthesis during brief exposures to 10 mM glucose.

According to our hypothesis, this elevates GP levels for longer after glucose removal and delays glucose clearance. In both cases the cells were exposed to extracellular glucose for 50 s. Compilation of traces in D illustrates once more that the final rate of glucose clearance is similar in all cases and that increased glycogen synthesis only delays glucose utilization. In summary, these data are consistent with removal of glucose causing a switch from glycogen synthesis to glycogen breakdown, thereby increasing GP which blocks the utilization of glucose by mitochondria-bound HK, in effect diverting GP to glycolysis.

In the presence of glucose, a large fraction of HKII was bound to mitochondria, and upon removal of extracellular glucose, HKII rapidly translocated to the cytosol with a time constant averaging 8. This effect was fully reversible, such that upon re-addition of glucose, HKII re-associated with mitochondria with a time constant of These data support the hypothesis that HKI binds strongly to mitochondria, while the distribution of HKII between cytoplasm and mitochondria is labile, dynamically regulated by glucose availability.

Panel C shows the rates measured as ratio of fluorescence intensity obtained from intracellular domains without and with mitochondria.

In almost all cases the region without mitochondria was selected at the cell periphery and that with mitochondria was selected near the nucleus. C2 shows the rate of HKII reassociation with mitochondria after glucose readdition. To evaluate the functional consequences of HKII redistribution on glucose metabolism, cells were subjected to a 20 to 30 min pre-incubation in the absence of glucose to maximize dissociation of HKII from mitochondria Fig.

This finding demonstrates that the subcellular distribution of HKII strongly influences its ability to promote anabolic use of glucose for glycogen synthesis. Images in A obtained with overexpression of a constitutively active Akt illustrate how Akt prevents HKII dissociation evoked by removal of glucose. Panel B shows a quantification of this effect, comparing the rate of HKII dissociation from mitochondria in response to glucose removal in the presence and absence of exogenous Akt.

Data in Panel C show how glucose clearance is affected by preincubation in the absence of glucose and how constitutively active Akt prevents this effect. Previous data Fig. Thus, glucose-induced HK dissociation slows the rate of glucose utilization.

The recording in C3 obtained with cells overexpressing constitutively active Akt indicates that Akt prevents the decrease in glucose clearance induced by incubation in the absence of glucose for 30 min. The bar graph in C4 quantifies the effects of glucose removal and Akt on glucose clearance. Since Akt facilitates HK interaction with mitochondria [14] , we examined whether translocation of HKII from mitochondria to cytoplasm in response to zero glucose pre-incubation was suppressed by overexpression of constitutively activate Akt.

Thus, both glucose and Akt signaling promote the binding of HKII to mitochondria, favoring glucose catabolism over glycogen synthesis. We tested this hypothesis using both direct and indirect approaches. First, in the presence of glucose, we applied iodoacetate IAA, 0.

As shown in Fig. These data support our hypothesis that the effect of glucose on HKII translocation and inhibition is mediated via GP. However when combined with our data obtained from intact cells, indicate that an intracellular factor is present in intact cells that prevents the dissociation of HKI induced by GP in isolated mitochondria.

Importantly, our data suggest that, in the absence of any factor other than GP the two HKs have similar affinity for mitochondrial membrane and dissociate at similar rates. The triangles represent measurements obtained with GP nM and the circles are for values obtained in the absence of GP.

Panel C3 shows the change in intracellular GP levels following glucose removal. Together, these data corroborate our glucose metabolism measurements, and support the hypothesis that upon glucose removal GP elevation inhibits glucose phosphorylation by hexokinases. The decay in GP that follows, accounts for the gradual reactivation of hexokinase activity and resumption of glucose phosphorylation. Catabolic and anabolic glucose utilization are both directed by hexokinases, which channel GP to glycolysis or glycogen and lipid synthesis.

While HKs coexist in many cell types, cells that generate glycogen in response to insulin, such as adult muscle, express primarily HKII, whereas cells that rely primarily on glycolysis for energy production, such as the brain, express high levels of HKI.

The specificity of these enzymes is not related to functional differences, since both phosphorylate glucose to GP, but may instead depend on subcellular location, reflecting spatial compartmentalization of glucose metabolism. Activation of the HK II gene by glucagon at first appears paradoxical based on our knowledge of the role of this hormone in normal tissues where it opposes the action of insulin.

Had the expression of a low K m HK been the only requirement of cancer cells, expression of HK I and HK III isozymes likely would have been observed as well, a phenotype that has not been encountered in studies to date. Therefore, it can be inferred that even at the terminal stages of cancer progression in the cachexic patient, the tumor will continue to harness glucose from the patient's body and thrive whereas the rest of the body systematically shuts down.

Finally, from the above discussion it seems clear that the presence of a multitude of cis -elements, or the lack thereof, between HK II and HK I promoters provides an explanation for the predominant expression of HK II in malignant tumors that exhibit the high glycolytic phenotype.

Therefore, the HK II promoter seems ideally tailored to provide an enhanced response to microenvironmental stimuli, resulting in greater HK II synthesis. In a given highly glycolytic tumor, a predominant fraction of the HK II is localized on the mitochondria with the enzyme anchored to the VDAC protein via an N-terminal-binding domain. Although once viewed only as a reason for metabolites like glucose to obtain preferred access to mitochondrial generated ATP, this HK—mitochondrial interaction is now believed to be a key component that regulates the cellular apoptotic signaling cascades that ultimately decide the fate of a tumor, as well as that of the host.

Akt in turn is activated by the upstream phosphoinositide 3-kinase PI 3-kinase pathway, which is stimulated by growth factor signaling. As illustrated in Figure 3 , Akt is also known to be a potent effector of antiapoptotic stimuli in tumors Gottlob et al. Mitochondrial-bound hexokinase HK II plays a major role in preventing tumor apoptosis.

Right: Without control mechanisms in place to prevent it, cell death would be highly likely within the unfavorable conditions that exist in a tumor microenvironment. Thus, caspase-mediated induction of apoptosis would be facilitated first by activation of the mitochondrial permeability transition pore complex MPTP , indicated on the right by a question mark?

This in turn inhibits access of VDACs to Bax and Bad, and most likely maintains cytochrome c in a state favorable for its mitochondrial retention in the inter-membrane space. Thus, HK II helps assure a highly malignant tumor's proliferation, and its escape from cell death, under conditions that would otherwise favor this process. The authors recognize that some aspects of this figure remain open to discussion and will necessitate additional studies to verify, modify or negate.

Voltage-dependent anion channel-bound HK predominantly HK II in cancers is thought to prevent apoptosis via several mechanisms whereby the formation of the mitochondrial permeability transition pore complex MPTP is inhibited. In fact, secondary disruption of the HK—VDAC interaction via non-Akt involved pathways, even in the absence of activation of proapoptotic factors such as Bax and Bak, induces apoptosis Majewski et al. The proapoptotic factors Bax and Bak are activated by their upstream regulator tBid, a proteolytically processed truncated form of Bid, another of the apoptotic family of proteins Wei et al.

Finally, ectopic expression of only the N-terminal domain of HK II, which alone can maintain its catalytic activity and contains the mitochondrial-binding domain, can still antagonize tBID Majewski et al. A recent study has also indicated that in liver cells a complex between glucokinase the high K m isozyme among HKs that is predominantly expressed in the liver, and long known to be cytoplasmic and Bad exists that is bound to the mitochondria Danial et al. Thus a glucokinase—Bad—VDAC interaction may indicate a role for Bad in integrating pathways of glucose metabolism and apoptosis in liver tissue.

However, a more recent report challenges this view of a glucokinase-mitochondrial interaction, as the latter authors have been unable to identify mitochondria-bound glucokinase Bustamante et al. Thus, more studies are needed to clarify this novel association and its implied metabolic consequences. The primary initializing event during induction of cellular apoptosis is the alteration in permeability of the mitochondrial membranes, which cause the release of cytochrome c from the apical surface of the mitochondrial inner membrane into the cytoplasm.

Released cytochrome c activates cellular caspases resulting in apoptotic cell death. Members of the BcL-2 family of proteins, which are either antiapoptotic e. Bcl, Bcl-X L or proapoptotic e. When HK is released from VDAC via manipulation of glucosephosphate levels or with compounds that disrupt the VDAC—HK interaction, tumor cells rapidly undergo apoptosis under a variety of stimuli which were previously ineffective in inducing apoptosis Pastorino et al.

Thus, occupation of VDAC by HK may initiate a series of molecular changes in key proteins in the inner mitochondrial membrane that, in turn, prevent the creation of a MPT pore complex.

Thus, the exact role of these translocators in facilitating apoptosis remains to be elucidated Kokoszka et al. As implied above, malignant tumors are defined by their uncontrolled proliferative capacity and their resistance to apoptosis.

This in turn will result in altered conformations in the channel proteins as well as disruption of optimal glycolytic energy generation. Such strategies may involve direct inhibition of the synthesis of HK isoforms. Additionally, what might be employed are small-molecule drugs such as Lonidamine Fanciulli et al.

In animal models, the use of a halogenated pyruvate derivative 3-bromopyruvic acid , has shown high efficacy against advanced stage malignant tumors Ko et al. In fact, in the most recent report, advanced stage cancers were eradicated by 3-bromopyruvate in 19 out of 19 animals without any recurrence Ko et al. Hexokinase—mitochondrial interactions, once considered only necessary to facilitate and maintain a highly glycolytic rate in malignant tumors, have now been shown to be crucial also for tumor survival.

Thus, mitochondrial-bound HK, now recognized as acting as both a facilitator and gatekeeper of the malignant state in many cancers will by necessity become one of the primary targets of those focused on eradicating not one or even a few cancer types, but all cancers that are shown clinically to be PET scan positive. J Biol Chem : — J Bioenerg Biomembr 24 : 47— Biochem J : — Biochem Cell Biol 80 : — Bustamante E, Pedersen PL.

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