Hexokinase kinetic studies

Because ATP and GTP are required in many biosynthetic and mechanochemical reactions, the ability to manipulate their concentrations is particularly important in many kinetic studies. The three enzymes most commonly employed for this purpose are hexokinase, phosphofruc-tokinase, and alkaline phosphatase. Each of these enzymes offers advantages and disadvantages, and one must consider the design requirements for any particular experiment before using one of these to deplete ATP or GTP. 

Hexokinase provides another example of the stereochemical inferences that can be drawn on the basis of kinetic studies with alternative substrates and competi-... 

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... 

This model has been reinforced by kinetic studies. The five-carbon sugar xylose, stereochemically similar to glucose but one carbon shorter, binds to hexokinase but in a position where it cannot be phosphorylated. Nevertheless, addition of xylose to the reaction mixture increases the rate of ATP hydrolysis. Evidently, the binding of xylose is sufficient to induce a change in... 

From pH kinetic studies, Viola and Cleland have proposed that hexokinase requires a group on the enzyme that must be unprotonated for the forward... 

Structural studies of the oxy-Cope catalytic antibody system reinforce the idea that conformational dynamics of both protein and substrate are intimately intertwined with enzyme catalysis, and consideration of these dynamics is essential for complete understanding of biologically catalyzed reactions. Indeed, recent single molecule kinetic studies of enzyme-catalyzed reactions also suggest that different conformations of proteins are associated with different catalytic rates (Xie and Lu, 1999). In addition, a number of enzymes are known to undergo conformational changes on binding of substrate (Koshland, 1987) that lead to enhanced catalysis two examples are hexokinase (Anderson and Steitz, 1975 Dela-Fuente and Sols, 1970) and triosephosphate isomerase (Knowles, 1991). 

Coe and Bessell and coworkers studied the metabolic fates of 2-deoxy-2-fluoro-D-glucose (2DFG) and related compounds by using yeast hexokinase (as a model for mammalian hexokinase), and determined the kinetic constants K and V ) of the Michaelis-Menten equation D-glucose 0.17 (K in mAf)> 1 00 (relative value, D-glucose taken as 1) 2DG 0.59 0.11, 0.85 2DFG 0.19 0.03, 0.50 2-deoxy-2-fluoro-D-mannose (2DFM) 0.41 0.05, 0.85 2-deoxy-2,2-difluoro-D-nraZ>//Jo-hexose... 

For example, Bachelard used [Mgtotai]/[ATPtotai ] = 1 in his rate studies, and he obtained a slightly sigmoidal plot of initial velocity versus substrate ATP concentration. This culminated in the erroneous proposal that brain hexokinase was allosterically activated by magnesium ions and by magnesium ion-adenosine triphosphate complex. Purich and Fromm demonstrated that failure to achieve adequate experimental control over the free magnesium ion concentration can wreak havoc on the examination of enzyme kinetic behavior. Indeed, these investigators were able to account fully for the effects obtained in the previous hexokinase study. ... 

This enzyme plays a key role in the metabolism of glucose and other related sugars. The physical and kinetic properties of yeast hexokinase have been extensively studied. Numerous recent studies have been made of its role in the phosphoryl transfer reaction. 

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. 

Hexokinase does not yield parallel reciprocal plots, so the Ping Pong mechanism can be discarded. However, initial velocity studies alone will noi discriminate between the rapid equilibrium random and steady-state ordered mechanisms. Both yield ihe same velocity equation and families of intersecting reciprocal plots. Other diagnostic procedures must be used (e.g., product inhibition, dead-end inhibition, equilibrium substrate binding, and isotope exchange studies). These procedures are described in detail in the author s Enzyme Kinetics behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Wiley-Interscience (1975),... 

The Kf for glucokinase, a liver-specific enzyme, is 20 mM. (We shall use the expression here, even though some hexokinases studied do not follow Michaelis-Menten kinetics, and the term [S]q3 might be more appropriate. Not aU enzymes have a but they do all have a substrate concentration that gives rise to V /2. 

Once the nature of the phosphoamino add is established, its catalytic competence has to be demonstrated. This can be done by kinetic experiments demonstrating the existence of double-displacement (or ping-pong) kinetics. However, if sequential kinetics is observ it will have to be own that the rates of the partial reactions leading to phosphorylation or dephosphorylation are comparable to the overall catalytic rate (Bridget et al., 1968 Wimmer and Rose, 1978). Not in all cases where a phosphoamino acid was identified could this condition be fulfilled, as is demonstrated clearly by penetrating studies on hexokinase (Wimmer and Rose, 1978). 

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