Phosphoryl transfer hexokinase

Figure 10.3 In the phosphoryl-transfer reaction catalysed by hexokinase, the y-phosphoryl group of ATP undergoes inversion of configuration. (From Voet and Voet, 2004. Reproduced with permission from John Wiley Sons., Inc.)...

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. 

Much of the earlier work on hexokinase has been summarized in two reviews that appeared in 1973 (25,26). The emphasis here will be on information that has appeared since that time. Significant advances have been made with respect to kinetic mechanism, stereospecificity of phosphoryl transfer, and x-ray structure. 

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). 

Hexokinase demonstrates the induced fit phenomenon it catalyzes phosphoryl transfer from ATP to C-6 of glucose as follows ... 

Phosphoryl transfer is a fundamental reaction in biochemistry and is one that was discussed in mechanistic and structural detail earlier (Section 9.4). Kinases are enzymes that catalyze the transfer of a phosphoryl group from ATP to an acceptor. Hexokinase, then, catalyzes the transfer of a phosphoryl group from ATP to a variety of six-carbon sugars (hexoses), such as glucose and mannose. Hexokinase, like adenylate kinase (Section 9.4.2) and all other kinases,... 

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. 

Induced fit can therefore only be used to explain the rates of reactions of very poor substrates compared with very good ones, e.g. the rate of phosphoryl transfer to water compared with that to glucose catalysed by hexokinase. Although water can almost certainly bind to the active site it must have insufficient binding energy to induce the necessary conformational change in the enzyme. 

The detection and characterization of intermediates in complex reactions is often accomplished through the use of isotope-exchange studies. In the case of enzymic phosphoryl transfer reactions, the presence of a phosphorylated enzyme intermediate is implicated by a partial exchange process as exemplified by the following reaction of hexokinase (Knowles, 1980) ... 

Isotopic labeling studies of phosphotranferase reactions culminated in the synthesis of ATP chiral at the y-phosphorus. Chirality was achieved by the synthesis of [y- 0, 0, 0]ATP of one configuration, and the analysis of its chirality was achieved by stereochenucally controlled transfer of the y-phosphoryl moiety to (S)-propane-l,2-diol where the absolute configuration was determined by a chemical/mass spectrometric sequence. The observation of inversion of configuration has been accepted as evidence of an in-line displacement mechanism at phosphorus by the two bound substrates the observation of retention of configuration was used to implicate the existence of a phosphoryl enzyme intermediate in the phosphoryl transfer process. For hexokinase, our case study, the finding is one of inversion, consistent with a direct transfer mechanism. 

The use of thiophosphate analogs of biophosphates in studying stereochemical problems was first introduced by Eckstein (1975) and subsequently widely applied to various problems. To illustrate the use of chiral thiophosphates in stereochemistry, consider the phosphoryl transfer reaction catalyzed by hexokinase (Scheme 1). Three types of problems can be studied by... 

This reaction, which is irreversible under intracellular conditions, is catalyzed by hexokinase. Recall that kinases are enzymes that catalyze the transfer of the terminal phosphoryl group from ATP to an acceptor nucleophile (see Fig. 13-10). Kinases are a subclass of transferases (see Table 6-3). The acceptor in the case of hexokinase is a hexose, normally D-glucose, although hexokinase also catalyzes the phosphorylation of other common hexoses, such as D-fructose and D-mannose. 

As enzymic reactions are in theory reversible, an analogue of a nucleoside triphosphate (e.g. I or II) may be formed in which the S,Y phosphoryl residues of the triphosphate moiety are replaced by PAA or PFA residues. We have prepared the ATP analogue of PAA. This compound has, not unexpectedly, no effect on enzymes such as hexokinase which transfer the Y phosphoryl residue of ATP to a substrate. This ATP analogue is also not a substrate RNA polymerase from E. aoli or from influenza virus. Furthermore, the dTTP analogue is not a substrate for the DNA polymerase of HSV (7). 

---