Although several isoforms of VDAC are known, they do have similar kinetic characteristics, which indicate that the contribution of VDAC to enhanced HK binding and glucose phosphorylation is due to quantitative differences in binding site availability Shinohara et al.
Most accessory proteins that modulate apoptosis via their interactions with mitochondria also use VDAC-1 as their anchor. In the latter case, the opened VDAC is proposed to restore metabolic exchange across the outer mitochondrial membrane while preventing the release of cytochrome c Vander Heiden et al. With regard to the enhanced glycolysis in tumors, an alternate view has also been put forward, where suppression of mitochondrial function owing to closure of porins is proposed to be responsible Lemasters and Holmuhamedov, According to this hypothesis, HK binding to VDAC inhibits its conductance and thus suppresses mitochondrial function while stimulating glycolysis.
However, this view makes it difficult to explain the well-observed phenomenon of direct access of mitochondrial generated ATP to the VDAC-bound HK Arora and Pedersen, , and thus needs further examination. The respective HK isozymes and transporter isoforms that are implicated in enhancing glucose phosphorylation with concomitant upregulation of glycolysis in malignant tumors are encoded at different chromosomal loci.
Tumors harness a multitude of genetic, epigenetic, transcriptional and post-translational strategies for enhanced expression and function of hexokinase HK II. During tumorigenesis of tissues where HK II is absent, the gene may be first brought out of its hibernation by demethylation, and then amplified 5—fold. Subsequently, the highly promiscuous promoter of the gene, which is activated by HIF-1, p53, glucose, and by both insulin and glucagon, further facilitates the tumor's requirements regardless of the nutritional status of the tumor-bearing host, and fuels the enhanced and continued synthesis of the gene product.
In contrast to other hexokinase isoforms, HK II harbors two active sites per enzyme moiety. As much as a fold amplification of the enzyme may be observed in malignant tumors owing to these different processes. Recently, epigenetic events leading to activation or silencing of alternate HK gene isoforms have been described during hepato-carcinogenesis.
Methylation restriction endonuclease analysis of normal hepatocytes and hepatoma cells has indicated differential methylation patterns in the HK II gene promoter during tumorigenesis where the HK isozyme expression shifts from HK IV, that exhibits a low affinity for glucose, to the HK II and HK I that exhibit a high affinity for glucose Goel et al.
Thus, these observations indicate that one of the initial events in activating the HK II gene during either transformation or tumor progression may reside at the epigenetic level. A first indication that gene amplification plays a role in enhanced expression of HK isozymes with a low K m for glucose high apparent affinity during tumorigenesis was demonstrated with Southern blot analysis and fluorescence in situ hybridization of hepatocytes and a hepatoma cell line.
Here, enhanced HK II expression was observed to be associated with at least a fivefold amplification of the HK II gene relative to that of normal hepatocytes Rempel et al. This gene amplification was located intra-chromosomally, and most likely occurs at the site of the resident gene.
No rearrangement of the gene was detected. Thus, these findings revealed that gene amplification plays a key role in overexpression of the low K m high apparent affinity HK II isoform in a highly malignant tumor expressing the high glycolytic phenotype.
Whether this will prove to be the case in other highly malignant tumors, or whether such tumors have devised multiple strategies for assuring enhanced glucose utilization, remains to be established. Evidence for the involvement of enhanced transcription of HK II in a malignant tumor expressing the high glycolytic phenotype came first from mRNA analysis via Northern blots Johansson et al. Here, an approximately fold enhancement of the message was observed over the background mRNA signal.
Thus, these studies strongly implicated HK II promoter activation and upregulation during tumorigenesis. Significantly, functional response elements for hypoxia HIF-1 , and p53 were located on the distal region of the promoter with the proximal region also being signficantly implicated in the hypoxic response Mathupala et al.
As the exact cis -elements for glucose and insulin response are unknown, their precise locations on the HK II promoter remain to be identified. 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.
For additional information, see: Carbohydrate Metabolism. Mechanism of liver glucokinase. Mol Cell Biochem. PMID: Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase. PMID: doi Cell-specific roles of glucokinase in glucose homeostasis.
Recent Prog Horm Res. Massachusetts Institute of Technology. Category : Topic Page. Hexokinase From Proteopedia.
Jump to: navigation , search. Show: Asymmetric Unit Biological Assembly. Export Animated Image. The chain is folded into two distinct regions, a small and large domain. Glucokinase has one active binding site for glucose and one for ATP, which is the energy source for phosphorylation.
This active binding site is located between the small and large domains. The carboxyl terminus is part of the alpha 13 helix, which codes for the region that forms half of the binding site for glucose. Glucokinase can be modulated to form an inactive and active complex. The inactive conformation forms when the alpha 13 helix has been modulated away from the rest of the molecule forming a large space. This space is too large to bind glucose so it is said to be in the inactive form.
The alternative is when the alpha 13 helix is modulated to form a smaller space thus activating the protein [4]. Glucokinase includes the where glucose forms hydrogen bonds at the bottom of the deep crevice between the large domain and the small domain. E, E shown in green of the large domain, T, K shown in red of the small domain, and N, D shown in yellow of a connecting region form hydrogen bonds with glucose.
The shows a different conformation. The again shows structural differences. 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. The first three are called low-K isozymes due to their high affinity for glucose.
They are inhibited by their product glucosephosphate. Hexokinase I is present in all mammalian tissues and is not affected by most changes, whether physiological, hormonal or metabolic. Hexokinase II is the principal regulated isoform in many cell types, and is in the muscle and heart, as well as in the mitochondria outer membrane.
It is seen in increased amounts in many cancers. Less is known about hexokinase III and its regulatory characteristics but it is substrate-inhibited by glucose at physiologic concentrations. Hexokinase Mechanism Hexokinase IV in mammals, which is also called glucokinase, has different kinetics and functions compared to other hexokinases. When it translocates between cytoplasm and nucleus of liver cells is the location of the phosphorylation on a subcellular level occurs.
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