Hexokinase from yeast

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. 

The hexokinases from yeast or ascites tumor cells showed only a marginal preference for a-glucose. The Km values for a- and /3-glucose (58 and 66 mM for the yeast enzyme and 77 and 82 mM for the tumor enzyme) were essentially the same, and the turnover numbers were only 10-20% higher with a-glucose. 

Figure 34.16. Actin and Hexokinase. A comparison of actin (blue) and hexokinase from yeast (red) reveals structural similarities indicative of homology. Both proteins have a deep cleft in which nucleotides bind. 

The introduction of the invertase from yeast alone was not sufficient. Invertase cleaves sucrose to release the two component sugars, glucose and fructose. While fructose can be readily metabolized by fructokinase in potato tubers, there is insufficient hexokinase activity in developing potato tubers to bring the glucose into intermediary metabolism. Therefore, it was necessary to introduce a second transgene, a bacterial glucokinase, in order to ensure that the hexoses became available for subsequent metabolism.25... 

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)). 

Hexokinase 280 kU/l/glucose-6-phosphate dehydrogenase 140 kU/1 hexokinase and glucose-6-phosphate dehydrogenase from yeast, suspended in 3.2 M ammonium sulfate solution. Dilute stock solutions with 3.2 M ammonium sulfate solution according to specified activity to 280 kU/1 and 140 kU/1, respectively. 

Fig. 17. A schematic drawing of the course of the polypeptide backbone of yeast hexokinase. From Anderson et al. (74). Reprinted with permission of Academic Press.

D-Glucosamine is phosphorylated by a hexokinase preparation from yeast. The resulting phosphorylated derivative consumes four moles of periodate per mole and is, therefore, considered to be D-glucosamine 6-phosphate. 

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... 

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... 

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... 

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...

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. 

The x-ray structure determination of yeast hexokinase has been undertaken by the Steitz group at Yale University (68). Although the complete amino acid sequence of hexokinase has not been determined, much information about the structure and function of this enzyme has been determined from the x-ray picture presented thus far. 

Schematic representation of the change in conformation of the hexokinase enzyme on binding substrate. E and E are the inactive and active conformations of the enzyme, respectively. G is the sugar substrate. Regions of protein or substrate surface excluded from contact with solvent are indicated by a crinkled line. Figure 8.3 presents a more detailed view of the hexokinase molecule. (Source From W. S. Bennett and T. A. Steitz, Glucose-induced conformational changes in yeast hexokinase, Proc. Natl. Acad. Sci. USA 75 4848, 1978.)...

---