Hexokinase yeast

Steitz, T.A., et al. High resolution x-ray structure of yeast hexokinase, an allosteric protein exhibiting a non-symmetric arrangement of subunits. 

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... 

Deoxy-3-fluoro-D-glucose (see Section 11,2), a weak substrate for yeast hexokinase, is phosphorylated enzymically - to give the 6-phosphate 588, which is transformed into 2-deoxy-2-fluoro-D-arabinose 5-phos-phate (589) by lead tetraacetate oxidation. 

The phosphorylation of D-glucosamine (2-amino-2-deoxy-D-glucose) was first achieved by incubating it with brain extracts and ATP.266 Crystalline, yeast hexokinase plus ATP also phosphorylates D-glucosamine, to give the 6-phosphate, at a rate at least 75 % of that of phosphorylation of D-glucose ... 

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... 

The observation that hexokinase preparations from ox retina react with D-glucose and D-fructose but not with 3-methyl D-glucose (M. Kerly, Biochem. J., 42, xx (1948)) has no bearing on this question, since animal hexokinase differs from yeast hexokinase (see Gerti T. Cori and M. W. Slein, Federation Proc., 6, No. 1 (1947)). 

Treatment of 9-(/ -D-ribofuranosyluronic acid)adenine with diphenylphosphoro-chloridate and orthophosphate or tripolyphosphate yields (62) and (63), which, although unstable, inhibit rabbit AMP aminohydrolase and pyruvate kinase, respectively, with behaviour characteristic of active-site-specific reagents.98 Adenylate kinases from several sources are inactivated by iV6-[2- and 4-fluorobenzoyl]-adenosine-5 -triphosphates, with kinetics characteristic of active-site labelling, although these compounds were without effect on yeast hexokinase and rabbit pyruvate kinase.99... 

NMR properties, 33 213, 274 partitioning, yeast hexokinase, 28 343, 344 tracer measurements, 32 320... 

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. 

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. 

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.

Figure 2. Hypothetical scheme for the selectivity-conferring interactions (indicated as wavy lines) of hexokinase with keto- and ald-hexoses. This model accounts for all known specificity and selectivity of yeast hexokinase toward hexose substrates and pentose ligands.

The reacting enzyme centrifugation technique has been used with yeast hexokinase and with phosphoenolpy-ruvate carboxykinase. ... 

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) ... 

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

Some enzymes, such as yeast hexokinase and creatine kinase (Chapter 12), associate in extremely asymmetric ways.102 A dimer is formed by means of heterologous interactions but steric hindrance prevents the unsatisfied sets of interacting groups from joining with additional monomers to form higher polymers. 

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. 

Hexokinase has a molecular weight of 102,000 and is composed of two identical subunits of 51,000 molecular weight each (20-22). In yeast, hexokinase exists as a mixture of two isoenzymes (23). These forms have been named A and B (23). These isoenzymes can be separated by chromatography and have been found to be chemically different (23, 24). Some of the earlier work with hexokinase was done with preparations that contained a mixture of isoenzymes. Work also was done with enzyme that was proteolytically modified. These variations undoubtedly have been responsible for some of the controversy concerning the properties of this enzyme. 

Kinetic Constants for the Sugar Substrates of Yeast Hexokinase... 

Fig. 10. Structures of sugars and sugar analogs that have been tested as substrates for yeast hexokinase.

Cornelius and Cleland have recently determined the absolute stereochemistry of the Mg2+ complex of ATP that is active with yeast hexokinase (43). MgATP can exist in solution as a pair of bidentate diastereomers (monodentate and tridentate complexes can also exist). The two possible bidentate isomers are shown schematically in Fig. 11. The two configurations have been labeled A and A (44). There are also four possible tridentate isomers. 

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 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. 

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