Sugar substrates, phosphorylated

Of the protein kinases, protein kinase A is the best investigated and characterized (review Francis and Corbin, 1994). The functions of protein kinase A are diverse. Protein kinase A is involved in the regulation of metabolism of glycogen, lipids and sugars. Substrates of protein kinase A may be other protein kinases, as well as enzymes of intermediary metabolism. Protein kinase A is also involved in cAMP-stimulated transcription of genes that have a cAMP-responsive element in their control region (review Montminy, 1997). An increase in cAMP concentration leads to activation of protein kinase A which phosphorylates the transcription factor CREB at Ser 133. CREB only binds to the transcriptional coactivator CBP in the phosphorylated state and stimulates transcription (see Chapter 1.4.4.2). 

Three types of lyases have been identified that catalyze the addition of phosphoenolpyruvate (PEP) to aldoses or to terminally phosphorylated sugar derivatives. With simultaneous release of inorganic phosphate from the preformed enolpyruvate nucleophile during C-C bond formation the additions are essentially irreversible and, therefore, these lyases are often referred to as synthases. The mechanistic details of these reactions, however, have yet to be elucidated but it seems obvious that the chances of variation on the part of the nucleophile will be strictly limited. Although the thermodynamic advantage makes these enzymes highly attractive for synthetic applications, none of them is yet commercially available and only few data have been reported concerning the individual specificities towards aldehydic substrates. 

On the other hand, the enzymatic synthesis of glycoconjugates and oligosaccharides leads to high product yields in a short time by stereo- and regioselective one-step reactions. All enzymatic reactions are easy to scale up and are carried out in aqueous media under mild conditions. A whole set of enzymes is now available to build up OAT bonds in monosaccharides, COP bonds in activated monosaccharides e.g. phosphorylated sugars or nucleotide sugars, and C-O-C bonds in di- and oligosaccharides (Fig. 1). However, all these enzymatic reactions are limited by the substrate spectrum of the individual enzyme. 

Proportional to this oxidation energy can and must be stored by phosphorylation of carboxylate and sugar substrates which is done by Mg or Zn enzymes (kinases, phosphatases) mainly. 

Random mechanisms wiU not show substrate inhibition of exchanges unless the levels of reactants that can form an abortive complex are varied together. The relative rates of the two exchanges will show whether catalysis is totally rate limiting (a rapid equilibrium random mechanism), or whether release of a reactant is slower. For kinases that phosphorylate sugars, the usual pattern is for sugar release to be partly rate limiting, but for nucleotides to dissociate rapidly (15, 16). 

Besides fueling biosynthetic processes, ATP hydrolysis also serves to spark many metabolic reactions. Thus, phosphorylation of substrates - sugars, for example -often initiates their metabolic transformation. In addition, a number of receptor-mediated, regulatory mechanisms depend on the splitting of ATP (or GTP). 

The phosphorylated sugar is used as a substrate for glycolysis to generate two moles of PEP, one of which is used to phosphorylate a further sugar molecule and the other to produce ATP (Figure 36.3). 

Dha systems of the PTS have three protein constituents, DhaM (IIA), DhaL (IIB), and DhaK (nC) (Siebold et al. 2003). DhaL corresponds in sequence to the A -termini of DHA kinases while DhaK corresponds to the C-tamini of these kinases. DhaM contains a domain that is distantly related to HA proteins of the mannose (Man) systems (family 2, Figure 4.2b) and can be fused to other PTS domains. Like the GAT systems, these systems are not full-fledged PTS systems because (i) DhaL contains tightly bound ADP which is phosphorylated rather than a histidyl or cysteyl residue in the proteins, and (ii) DhaK binds DHA covalently via a histidyl residue to provide specificity (Siebold et al. 2003 Garcia-AUes et al. 2004). Covalent bond formation between enzymes and the sugar substrate is not obsCTved for any other PTS enzyme. 

Enzyme preparations from liver or microbial sources were reported to show rather high substrate specificity [76] for the natural phosphorylated acceptor d-(18) but, at much reduced reaction rates, offer a rather broad substrate tolerance for polar, short-chain aldehydes [77-79]. Simple aliphatic or aromatic aldehydes are not converted. Therefore, the aldolase from Escherichia coli has been mutated for improved acceptance of nonphosphorylated and enantiomeric substrates toward facilitated enzymatic syntheses ofboth d- and t-sugars [80,81]. High stereoselectivity of the wild-type enzyme has been utilized in the preparation of compounds (23) / (24) and in a two-step enzymatic synthesis of (22), the N-terminal amino acid portion of nikkomycin antibiotics (Figure 10.12) [82]. 

Fig. 6. Vectorial phosphorylation by a mechanism in which translocation and phosphorylation of the sugar are two distinct steps. The product binding site of the translocator T (domain C of II ") would be the substrate binding site of the kinase K (domains A and B). Since both the left-hand cycle and the right-hand cycle are catalyzed by the same enzyme they will very likely be kinetically dependent. Note that the kinetic cycle on the left-hand side of the figure is identical to Fig. 5.

The emphasis in kinetic studies of E-IIs has been on the analysis of the rates of phosphorylation of the sugar by the phosphoryl group donor. In the early studies the question was addressed whether phosphorylated E-II would be a catalytic intermediate in the reaction or whether the phosphoryl group would be transferred directly from the donor to the sugar on a ternary complex between the enzyme and its substrates [66,75,95-100]. This matter has been satisfactorily resolved by a number of other techniques in favor of the first option and possible reasons why some systems did not behave according to a ping-pong type of mechanism have been discussed [1]. 

The most straight-forward interpretation of the phosphorylation reaction would be that exactly the same reaction is catalyzed as during transport of the sugar into the cell where the substrate is offered to the periplasmic side of the membrane (Eq. (1), overall reaction) phosphorylation would measure transport as well. However, this may not be the case several lines of evidence discussed in the previous sections indicate that the mechanism underlying the phosphorylation reaction could be much more complex. Factors that may complicate the interpretation of the phosphorylation reaction in detergent solutions are ... 

The first such case was concerned with the limited substrate acceptance of d-2-keto-3-deoxy-6-phospho-gluconate (KDPG) aldolase (161). This catalyzes the (reversible) reaction of pyruvate (41) to certain chiral aldehydes such as 42, with formation of aldol products such as 43. It was known that this aldolase is highly specific for chir l-phosphorylated aldehydes with the d configuration at the C2 position leading stereoselectively to a precursor of the corresponding d sugar such as 44 (162) ... 

---