Hexokinases HK

Although interest in inhibition of glycolytic enzymes as anticancer treatment had waned somewhat in the late 1990s, the description of the promising in vivo activity of 3-bromopyruvate (2), an inhibitor of hexokinase II, has prompted a renewed interest in this field. Efficacy of 3-bromopyruvate was reported against various cancer cell lines in animal studies at Johns Hopkins.11 Further investigation has revealed that direct intra-arterial administration to the site of the primary tumor may be especially effective.12,13 Despite the promising pre-clinical activity of 3-bromopyruvate, no human clinical trials have been reported. 

---

Figure 30. A medium complexity model of yeast glycolysis [342], The model consists of nine metabolites and nine reactions. The main regulatory step is the phosphofructokinase (PFK), combined with the hexokinase (HK) reaction into a single reaction vi. As in the minimal model, we only consider the inhibition by its substrate ATP, although PFK is known to have several effectors. External glucose (Glc ) and ethanol (EtOH) are assumed to be constant. Additional abbreviations Glucose (Glc), fructose 1,6 biphosphate (FBP), pool of triosephosphates (TP), 1,3 biphosphogly cerate (BPG), and the pool of pyruvate and acetaldehyde (Pyr).

A good example of allosteric inhibition is given by hexokinase (HK) isoenzymes of muscle. The product of the HK reaction, glucose-6-P allosterically inhibits the enzyme, so matching the phosphorylation of glucose to its overall metabolism, helps to regulate... 

Consider the case of glucose (Glc) and ATP interactions with hexokinase (HK) in the absence of catalysis (which can be accomplished by omitting magnesium ion). 

The elegant enzymatic procedure of Drawert and Kupper (59) permits determination of glucose, fructose, and (when necessary) sucrose. Glucose reacts with adenosintriphosphate (ATP) in the presence of hexokinase (HK) to produce glucose-6-phosphate. 

Fig. 9. Sequence of transformations catalysed by the supramolecular ATP-generating system [38, AcP, Mg2, ADP] (38 = [24]-N6C>2) and the enzymes hexokinase (HK), glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phospho-gluconate dehydrogenase (6-P-GDH) [5.32],...

Glucose and fructose are phosphorylated in the presence of adenosine triphosphate (ATP) in a reaction is catalysed by hexokinase (HK) producing respectively, glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) ... 

The ATP produced is measured by hexokinase (HK)/ glucose-6-phosphate dehydrogenase (G6PD) coupled reactions that ultimately convert NADP to NADPH, which is monitored spectrophotometricaUy. Oliver first reported this method that RosaUd also described with the improvement of adding AMP to inhibit adenylate kinase (AK) and cysteine to activate CK. Subsequently, Szasz and colleagues optimized the assay by adding N-acetylcysteine to activate CK, EDTA to bind Ca and to increase the stability of the reaction mixture, and adenosine pentaphosphate (ApsA) in addition to AMP to inhibit AK. A reference method based on this previous experience was developed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) it was modified recently to produce a reference procedure for the measurement of CK at 37 °C. ... 

Hexokinase (HK EC 2.7.1.1) catalyzes the phosphorylation of glucose to glucose-6-phosphate (G6P) using ATP as a phosphoryl donor. The activity of HK is significantly higher in reticulocytes compared with mature red cells, where it is very low. The HK reaction is one of two rate-limiting steps in this pathway, the other being the phosphofructokinase reaction. 

ATP-dependent hexokinase (HK, EC 2.7.1.1) converts both glucose anomers. Its Km for glucose is 10 mmol/1. The tetrameric protein (MW 100 000) contains eight SH-groups per molecule, of which four are essential for the enzyme s function. Owing to the high specificity of HK, glucose determination by means of this enzyme has become the inter-... 

Pig. 83. Principle of a sensor containing hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6P-DH) for determination of NADP+, ATP, glucose-6-phosphate, and fructose. 

The LOD-LDH thermistor has been combined with a reactor containing coimmobilized hexokinase (HK) and pyruvate kinase (PK) (Kirstein et al., 1987, 1989). These enzymes carry out the recycling of ATP/ADP whereby pyruvate is formed, which is then recycled in the LOD-LDH system. Although the enthalpy of the PK reaction is positive, the sufficiently negative enthalpies of the other three enzyme reactions provide an amplification factor of 1700 and thus a detection limit for ADP of 10 nmol/L. A PK-HK thermistor alone provided a 30-fold amplification and a detection limit of 2 pmol/1. 

Fig. 4.7.7. Chromatography of a crude yeast extract on Cibacron Blue F3G-A silica with on-line monitoring of hexokinase (HK) and 3-phosphoglycerate kinase (PGK) activity. Sample applied at first arrow crude yeast extract (2 pi) column irrigant, 0.1 M Tris-HCI buffer (pH 7.3) containing O.S mM EDTA, 5 mM MgClj and O.S mM 2-mercaptoethanol flow rates, I ml/min, both pumps post-column reactor temperature, 40 0.1°C assay mix temperature, 0°C temperature of analytical column, 22°C eluents (400 pi), as indicated by the arrows UV-detector (290 nm), 0.32 AUFS enzyme activity detector, 0.5 AUFS. Reproduced with permission from Ref. 126.

Fig. 21.13. Transport of compounds across the inner and outer mitochondrial membranes. The electrochemical potential gradient drives the transport of ions across the inner mitochondrial membrane on specific translocases. Each translocase is composed of specific membrane-spanning helices that bind only specific compounds (ANT adenine nucleotide translocase). In contrast, the outer membrane contains relatively large unspecific pores called VDAC (voltage-dependent anion channels) through which a wide range of ions diffuse. These bind cytosolic proteins such as hexokinase (HK), which enables HK to have access to newly exported ATP.

F i g. 5.11 Chemical reaction mechanism for an abbreviated model of the glycolytic pathway. The mechanism includes many of the known activations and inhibitions of enzymes by metabolites. Broken lines indicate activation (0) or inhibition (0) of enzymes by metabolites. The abbreviations for the enzymes are hexokinase (HK), phosphofrnctokinase (PFK), and pyruvate kinase (PK) the abbreviations for the five independent variables are fructose 6-phosphate (F6P), fructose 1,6-bisphosphate (FDP), phosphoenolpyruvate (PEP), pyruvate (PYR), and ATP (in fig. 5.12, these are labeled 1-5, respectively). (From [1].)... 

The reaction in which the substance to be determined is transformed is known as the auxiliary, while the reaction used for the actual measurement is called the indicator reaction. Both reactions can generally be carried out in a single assay mixture (coupled reactions). If a product of the auxiliary reaction is measured in the indicator reaction, one speaks of a succeeding indicator reaction. In this case, the function of the auxiliary enzyme is to convert the substance to be analyzed into a substance that can be measured in the indicator reaction. An example is the determination of glucose using hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6P-DH). 

Another interesting enzyme was chosen for the construction of a biosensor for ATP determination [161]. This is based on two enzymes (glucose oxidase and hexokinase-HK) competing for the same substrate (i.e., glucose) ... 

Tissue was homogenised with an equal volume of buffer (2 ml Tris-HCl (pH 8.0), 0.1 ml mercaptoethanol, 10 g sucrose, and 25 mg NADP per 100 ml H2O - adjusted to pH 7.2 with HCl). Electrophoresis was performed on cellulose acetate plates (Titan III Helena Laboratories). A preliminary survey of 42 enzymes detected four enzyme loci, coding for four different enzyme systems that were polymorphic and consistently resolvable. These enzymes were used for the analysis and were as follows (abbreviation and enzyme commission number in parentheses) hexokinase (HK, E.C. 2.7.1.1), mannose-6-phosphate isomerase (MPI, E.C. 5.3.1.8), 6-phosphogluconate dehydrogenase (6PGD, E.C. 1.1.1.44), and phosphoglucose isomerase (PGI, E.C. 5.3.1.9). 

---