Hexokinase mechanism

Retention, too, is highly tissue-specific. Sometimes, the extraction mechanism is also the retention mechanism, as for Tc-sestamibi, which is retained in mitochondria as long as transmembrane potentials remain intact. Others are separate. F-2-Fluorodeoxyglucose enters the cell by the same pathway as glucose, but is trapped because it is not a substrate for hexokinase, preventing further intracellular metabohsm. 

Mechanism for Gluconeogenesis. Since the glycolysis involves three energetically irreversible steps at the pyruvate kinase, phosphofructokinase, and hexokinase levels, the production of glucose from simple noncarbohydrate materials, for example, pyruvate or lactate, by a reversal of glycolysis ( from bottom upwards ) is impossible. Therefore, indirect reaction routes are to be sought for. 

The rate function vHK of the hexokinase reaction can, for example, be described by an irreversible random-order bireactant mechanism [101],... 

The key regulatory enzymes in glycolysis are phosphory-lase, hexokinase, phosphofructokinase and pyruvate kinase, the activities of which are stimulated by the increase in the concentrations of AMP and phosphate and the decrease in that of phosphocreatine. These mechanisms are discussed in Chapters 6 and 9 Figures 6.16 and 9.27. 

Rate experiments that are typically carried out in the presence of different concentrations of an alternative product (or product analog) while using the normal substrates . This approach can be particularly useful when the normal product cannot be used because it is unstable, insoluble, or ineffective (the latter indicated by a very high Ki value). Moreover, the normal product may be consumed as an essential substrate in a coupled assay system for the primary enzyme. Fromm and Zewe used the alternative product inhibition approach in their study of hexokinase. Wratten and Cleland later applied this procedure to exclude the Theorell-Chance mechanism for liver alcohol dehydrogenase. See Abortive Complexes... 

Haldane relationships can also be useful in characterizing isozymes or the same enzyme isolated from a different source. Reactions catalyzed by isozymes must have identical equilibrium constants, but the magnitudes of their kinetic parameters are usually different (e.g., the case of yeast and mammalian brain hexokinase ). Note that the Haldane relationship for the ordered Bi Bi mechanism is = Hmax,f p i iq/(f max.r ia b)- This same... 

For this reason, these alternative routes for isotope combination with enzyme-substrate and/or enzyme-product complexes ensures that raising the [A]/[Q] or [B]/[P] pair will not depress either the A< Q or the B< P exchanges. Fromm, Silverstein, and Boyer conducted a thorough analysis of the equilibrium exchange kinetic behavior of yeast hexokinase, and the data shown in Fig. 2 indicate that there is a random mechanism of substrate addition and product release. 

The results indicate that brain hexokinase does indeed operate by way of a sequential kinetic mechanism, and subsequent kinetic studies with reversible inhibitors support this conclusion. These comments reinforce the wisdom of remaining wary of mechanistic inferences drawn solely on the basis of initial rate studies alone. See also Initial Rate Enzyme Assays... 

OCTAHEDRAL COORDINATION HEXA UNI PING PONG ENZYME MECHANISM HEXOKINASE ATP GTP DEPLETION DEINHIBITION INDUCED FIT HYPOTHESIS HEXOSAMINIDASE A and B... 

The substrate specificities of both mammalian and yeast hexo-kinases have been extensively studied (76,77). Nevertheless, work in this area continues both in the search for isoenzyme specific inhibitors and in increasingly detailed investigations of the catalytic mechanism. Recently potential transition state analogs PI-(adenosine-5 )-P3-glucose-6 triphosphate (Ap -glucose) and P1-(adenosine-5 )-P4-glucose-6 triphosphate (Ap.-giucose) were tested as inhibitors of four hexokinase isoenzymes. However, they were found to exhibit less affinity for the enzyme than either of the natural substrates alone (78). 

The x-ray structure for yeast hexokinase is also available. Thus, glucose analogs are now being used to elucidate minute details of the catalytic mechanism. Recently D- lose was used in crystallographic work to show that the 6-hydro ethyl group of the natural substrate is necessary for substrate-induced closure of the active-site cleft (81 ). This induced closure, which is observed with glucose binding (82), is believed to be a part of the induced fit mechanism postulated for hexokinase (83) ... 

Induced fit is only one aspect of the catalytic mechanism of hexokinase—like chymotrypsin, hexokinase uses several catalytic strategies. For example, the active-... 

There is one case in which strain or induced fit could be useful in a type of specificity. These mechanisms are unimportant where competition between substrates is concerned. But given a situation in which there is no specific substrate present, these mechanisms could be of use in providing a low absolute activity of the enzyme toward, say, water. For example, induced fit could prevent hexokinase from being a rampant ATPase in the absence of glucose (although its absence is extremely unlikely). 

Much of the earlier work on hexokinase has been summarized in two reviews that appeared in 1973 (25,26). The emphasis here will be on information that has appeared since that time. Significant advances have been made with respect to kinetic mechanism, stereospecificity of phosphoryl transfer, and x-ray structure. 

In the last 15 years a large number of publications have been concerned with the determination of the kinetic mechanism of yeast hexokinase. Unfortunately, there has been much disagreement between various authors on the conclusions to be drawn from such studies. 

In a two-substrate reaction similar to that catalyzed by hexokinase, two basic mechanisms may be at work. First, a ping-pong reaction may be occurring in which the enzyme shuttles between a stable enzyme intermediate, such as a phosphorylated enzyme, and a free enzyme. Second, the reaction may be sequential, in which case no reaction occurs until both substrates are on the enzyme. There are two types of sequential mechanisms. If one substrate cannot bind until after the addition of the other substrate the mechanism is said to be ordered. However, if they can combine in any order the mechanism is said to be random. The various kinetic methods for distinguishing between these mechanistic forms have been summarized by Cleland (52). The evidence for and against these possible kinetic schemes will now be summarized for yeast hexokinase. 

In the absence of added glucose, hexokinase was found to catalyze the very slow hydrolysis of MgATP (59). This has been explained by assuming that water has replaced glucose at the active site of the enzyme. This ATPase activity can be inhibited by compounds that inhibit the hexokinase activity (60) and can be stimulated by compounds such as D-xylose or D-lyxose which lack the terminal -CH2OH of glucose (61). The ATPase reaction has been used to support evidence that hexokinase has a random kinetic mechanism, since it shows that ATP can bind to hexokinase in the absence of glucose (62). 

When taken collectively, the overall evidence indicates that hexokinase can both add and release substrates and products in a random mechanism. However, the mechanism cannot be described as rapid equilibrium random. The evidence also indicates that the preferred pathway is the ordered addition of glucose followed by ATP, then the release of ADP followed by glucose-6-P. Danenberg and Cleland have recently attempted to assign relative rate constants to a general random mechanism for hexokinase as shown in Fig. 14 (30). 

Fig. 18. A schematic drawing of the chemical mechanism of yeast hexokinase.

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