Yeast hexokinase reactions

The method of isotope trapping allows one to detemrine the stickiness of all substrates but the last to combine in an ordered mechanism and of all substrates in a random mechanism (Rose et al, 1974 Rose 1995). This method was developed initially by Rose for the yeast hexokinase reaction, and it is essentially a single turnover experiment in which one detemtines analytically the proportion of an enzyme-substrate complex that reacts to give products, as opposed to dissociating. 

As bacterial transglucosidase is instrumental in the transfer of a D-glucose residue from one acceptor to another, so does yeast hexokinase 3 catalyze a transphosphorylation. The highly specific donator of a labile phosphate group is adenosine triphosphate (XX), the fermentable hexoses D-glucose, D-mannose and D-fructose functioning as acceptors. Hexokinase catalyzes the reaction... 

Yeast hexokinase (1-2 international units per mL reaction solution) acting for 20 min in the presence of 10 mM D-glucose has proven to be quite effective in depleting ATP at levels not exceeding 1 mM. At pH 7 in the presence of 1-2 mM uncomplexed magnesium ion, the equilibrium constant for the hexokinase reaction is about 1500 thus, one can anticipate substantial conversion of 1 mM ATP, as indicated by the following equation ... 

GTP is also a substrate for yeast hexokinase, but phos-phofructokinase (PFK) acts on GTP more efficiently than hexokinase. Typically, one uses 1-2 international units PFK per milliliter of reaction solution (for 20 min in the presence of 5 mM o-fructose 6-phosphate) to deplete GTP initially present at concentrations up to 1 mM. 

Figure 2. (A) Effect of simultaneously raising the absolute concentrations of ATP and ADP on the indicated equilibrium exchange reactions catalyzed by yeast hexokinase. (B) Effect of simultaneously raising the absolute concentrations of glucose and glucose 6-phosphate on the indicated equilibrium exchange reactions of yeast hexokinase.

Yeast hexokinase (Mr 107,862) is a bisubstrate enzyme that catalyzes the reversible reaction... 

Figure 9-7 (A) Effect of glucose and glucose 6-phosphate concentrations on reaction rate of yeast hexokinase at equilibrium. Reaction mixtures contain 1-2.2 mM ATP, and 25.6 mM ADP at pH 6.5. From Fromm et al.51 (B) Effect of lactate and pyruvate concentrations on equilibrium reaction rates of rabbit muscle lactate dehydrogenase. Reaction mixtures contained 1.7 mM NAD+, and 30 - 46 pM NADH in Tris-nitrate buffer, pH 7.9, 25°C. From Silverstein and Boyer.53...

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. 

Which of the two possible isomers is active with yeast hexokinase Since Mg2 + exchanges ligands rapidly, it is not possible to separate the two species (45). Cornelius and Cleland synthesized Co(NH3)4ATP, which is bidentate and, since Co3+ exchanges ligands very slowly, the individual isomers should be stable to isomerization (46). Co(NH3)4ATP was found to be a substrate for hexokinase in the following reaction (43) ... 

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. 

D-Glucosamine 6-phosphate can be readily acetylated by a AT-acetylase obtained from a preparation of yeast hexokinase. The resulting iV-acetyl-D-glucosamine 6-phosphate is identified by its Morgan-Elson reaction. The acetyl-coenzyme A which appears to be required for this reaction may be generated by acetate, adenosine-5-triphosphoric acid, and coenzyme A (in the presence of an acetate-activating enzyme). 

The results summarized in Table III suggest that the enzymes that catalyze phosphoryl transfer via an inversion of configuration do so with an in-line transfer in a sequential mechanism. The mechanistic pathway is prevalent in the phosphokinases. Although this information does not provide direct evidence for an associative or Sn2 mechanism in contrast to a dissociative mechanism, if the latter process does occur then there is insufficient room at the catalytic site for the metaphosphate to rotate or dissociate and to cause racemization. The observation of a secondary 0 isotope effect less than 1.00 indicates that a dissociative transition state occurs with yeast hexokinase (57). The enzymes that demonstrate retention of configuration do so via double-displacement reactions. Mutases exclusively use this mechanistic pathway. 

The use of X-ray techniques to elucidate the three-dimensional structure of enzymes shows that many of them possess a characteristic concave cleft at the active site. Concavities of this type have been observed, for example, in the case of lysozyme [8, 9] trypsin [10], yeast hexokinase [11], liver alcohol dehydrogenase [12] and citrate synthase [13]. It is thus reasonable to assume that the interaction between an enzyme and its substrate, inhibitor or cofactor usually occurs not in bulk water but rather in a shielded proteic cleft whose specific microenvironment is induced by the amino acid residues forming the cleft. Hydrophobicity, electrostatics, solvation and a relatively low dielectric constant prevailing within the cleft no doubt play a decisive role in determining the nature and rate of the reaction catalyzed by the enzyme. 

As in so many reactions involving ATP, yeast hexokinase is activated by trace concentrations of a suitable divalent cation, such as magnesium or manganese. Despite this metal requirement, fluoride has no inhibitory effect. 

Fig. 15. Assay for P5C in biologic fluids. NADP[ H] was produced from [l- H]glucose in the presence of ATP and NADP in reactions catalyzed by yeast hexokinase and G-6-P dehydrogenase and subsequently separated from macromolecules by molecular sieving. Using NADPPH] thus produced, the production of PHjproline by a purified preparation of P5C reductase from E. colt (22) was a linear function of P5C in the assay. Adapted from ref. (30).

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).

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... 

Symbol for the temperature coefficient, a quotient equal to Vt+wIvt, where Vr+io and Vj are the rates of a process (e.g., an enzyme-catalyzed reaction) at two temperatures differing by 10°C. This parameter is usually evaluated at saturating concentrations of substrate(s), so that temperature-dependent changes in Michaelis constant(s) are inconsequential. The <2io value is a characteristic property of a particular enzyme from a specific organism and cell type. For example, one cannot use the Qio value for one hexokinase from yeast to infer the temperature dependence of another hexokinase, say from rat brain. Likewise, the Qio value need not remain the same for a mutant form and a wild-type enzyme. 

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