Hexokinase Conformation

The transporter binds glucose at a specific site, then changes its conformation, which results in transport of glucose across the membrane to be released on the other side. The enzyme hexokinase (or glucokinase) binds glucose at a specific site, then catalyses its phosphorylation and releases the product, glucose 6-phosphate. 

Initial rate kinetics of bovine brain hexokinase (A) with glucose and ATP as substrates, and (B) with fructose and ATP. Note that the apparent parallel-line kinetics observed with glucose conform to a rate equation lacking a < 12 term. 

ATP Triphosphate Chain Conformation. Much of the work in the area of ATP triphosphate chain conformation has been performed by Cleland and co-workers (14--16). Their studies on metal(III)ATP interactions with kinases have led to the classification of kinases according to the stereochemistry of the polyphosphate chain as it binds to the active site. For the kinases they studied (hexokinase, glycerokinase, creatine kinase, phosphofructokinase, 3-phosphoglycerate kinase, acetate kinase, arginine kinase, adenylate kinase and pyruvate kinase) it was found that B, y-bidentate chromi M(III)-ATP (CrATP) and not a,6,y-tridentate CrATP is a... 

FIGURE 6-22 Induced fit in hexokinase. (a) Hexokinase has a U-shaped structure (PDB ID 2YHX). (b) The ends pinch toward each other in a conformational change induced by binding of o-glucose (red) (derived from PDB ID 1HKG and PDB ID 1GLK). 

As mentioned, AMP-PNP or ADP in the presence of glucose will bind only to the BII crystals at a site between the two subunits. Nucleotides bound at this site appear to be in a fully extended conformation (73). ATP analogs bound at this site make contact with amino acid residues from both subunits. The y-phosphate of ATP bound at this site is 20 A from the 6-hydroxyl of bound glucose on one subunit and 30 A from the glucose on the other subunit (73). It has been proposed that this site is an allosteric regulatory site for hexokinase and not the substrate site for ATP where phosphoryl transfer occurs (73). 

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.)...

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). 

Figure 16.4. Induced Fit in Hexokinase. As shown in blue, the two lobes of hexokinase are separated in the absence of glucose. The conformation of hexokinase changes markedly on binding glucose, as shown in red. The two lobes of the enzyme come together and surround the substrate. [Courtesy of Dr. Thomas Steitz.]...

A. E. Aleshin, C. Kirby, X. Liu, G.P. Bourenkov, H.D. Bartunik, H.J. Fromm, and R.B. Honzatko. 2000. Crystal structures of mutant monomeric hexokinase I reveal multiple ADP binding sites and conformational changes relevant to allosteric regulation J. Mol. Biol. 296 1001-1015. (PubMed)... 

Hexokinase has a low ATPase activity in the absence of a sugar because it is in a catalytically inactive conformation. The addition of xylose closes the cleft between the two lobes of the enzyme. However, xylose lacks a hydroxymethyl group, and so it cannot be phosphorylated. Instead, a water molecule at the site normally occupied by the C-6 hydroxymethyl group acts as the acceptor of the phosphoryl group from ATP. 

Bennett, W. S., and Steitz, T. A. Glucose-induced conformational change in yeast hexokinase. Proc. Natl. Acad. Sci. USA 75, 4848-4854 (1978). 

McDonald, R. C., Steitz, T. A. and Engelman, D. M. (1979) Yeast Hexokinase in Solution Enhibits a Large Conformational Change upon Binding Glucose or Glucose 6-Phosphate, Biochemistry 18, 338-342. 

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