Synthesis of glucoconjugates of oleanolic acid as inhibitors of glycogen phosphorylase

Synthesis and biological evaluation of glucoconjugates of oleanolic acid, linked by either a triazole moiety or an ester function, as novel inhibitors of glycogen phosphorylase have been described. Several triterpene-glycoside conjugates exhibited moderate-to-good inhibitory activity against rabbit muscle GPa. Compound 12 showed the best inhibition with an IC₅₀ value of 1.14 μM. Structure-activity relationship (SAR) analysis of these inhibitors is also discussed. Possible binding modes of compound 12 were explored by molecular docking simulations.     

Fine structural properties of natural and synthetic glycogens

Glycogen, highly branched (1→4)(1→6)-linked α-d-glucan, can be extracted from natural sources such as animal tissues or shellfish (natural source glycogen, NSG). Glycogen can also be synthesized in vitro from glucose-1-phosphate using the cooperative action of α-glucan phosphorylase (GP, EC 2.4.1.1) and branching enzyme (BE, EC 2.4.1.18), or from short-chain amylose by the cooperative action of BE and amylomaltase (AM, EC 2.4.1.25). It has been shown that enzymatically synthesized glycogen (ESG) has structural and physicochemical properties similar to those of NSG. In this study, the fine structures of ESG and NSG were analyzed using isoamylase and α-amylase. Isoamylase completely hydrolyzed the α-1,6 linkages of ESG and NSG. The unit-chain distribution (distribution of degrees of polymerization (DP) of α-1,4 linked chains) of ESG was slightly narrower than that of NSG. α-Amylase treatment revealed that initial profiles of hydrolyses of ESG and NSG were almost the same: both glycogens were digested slowly, compared with starch. The final products from NSG by α-amylase hydrolysis were glucose, maltose, maltotriose, branched oligosaccharides with DP ? 4, and highly branched macrodextrin molecules with molecular weights of up to 10,000. When ESG was digested with excess amounts of α-amylase, much larger macrodextrins (molecular weight > 10⁶) were detected. In contrast, oligosaccharides with DP 4-7 could not be detected from ESG. These results suggest that the α-1,6 linkages in ESG molecules are more regularly distributed than those in NSG molecules.     

Cellobiose catabolism in the haloalkaliphilic hydrolytic bacterium <Emphasis Type="Italic">Alkaliflexus imshenetskii</Emphasis>

Cellobiose metabolism was studied in Alkaliflexus imshenetskii, a haloalkaliphilic hydrolytic bacterium capable of utilizing certain polymers of plant origin, as well as mono- and disaccharides. The major products of cellobiose fermentation by the bacterium were succinate and acetate, and formate was a minor product. Cellobiose could be split into glucose molecules by both β-glucosidase (hydrolytic pathway) and phosphorylase (phosphorolytic pathway); the activity of the former enzyme was two orders of magnitude higher (3600 nmol/(min mg) versus 36 nmol/(min mg)). In cell extracts of the bacterium, high activities of the Embden-Meyerhof-Parnas pathway enzymes—hexokinase, glucose-phosphate isomerase, and phosphofructokinase—were revealed, as well as the activities of glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and key enzymes of the Entner-Doudoroff pathway—6-phospho-gluconate dehydratase and 2-keto-3-deoxy-6-phospho-gluconate aldolase. Neither the activity of the key enzyme of the hexose-mono-phosphate pathway, 6-phospho-gluconate dehydrogenase, nor the activities of the key enzymes of the modified Entner-Doudoroff pathway, glucose dehydrogenase and 2-keto-3-deoxy-gluconate kinase, were revealed.     

Kinetic analysis and modelling of the allosteric behaviour of liver and muscle glycogen phosphorylases

Allosteric enzymes have very complex kinetic behaviours which are primarily interpreted through simplified models. To describe the functional properties of liver and muscle glycogen phosphorylase isozymes we have developed an experimental strategy based on the measurements of initial reaction rates in the presence of different concentrations of the effectors glucose-1-phosphate and methyl-xanthines. Using the extensive structural information available for the two glycogen phosphorylase conformers T (inactive) and R (active) with different ligands, we have applied the Monod-Wyman-Changeux model and analysed the results in the context of the exclusive binding of the inhibitors to the T state, meanwhile the substrate glucose-1-phosphate binds to both, the R and T states. The kinetic analysis shows a good agreement between our model and the results obtained from the glycogen phosphorylases and inhibitors included in this study, which demonstrates the validity of the approach described here.     

Structure of a mutant human purine nucleoside phosphorylase with the prodrug, 2‐fluoro‐2′‐deoxyadenosine and the cytotoxic drug, 2‐fluoroadenine

A double mutant of human purine nucleoside phosphorylase (hDM) with the amino acid mutations Glu201Gln:Asn243Asp cleaves adenosine‐based prodrugs to their corresponding cytotoxic drugs. When fused to an anti‐tumor targeting component, hDM is targeted to tumor cells, where it effectively catalyzes phosphorolysis of the prodrug, 2‐fluoro‐2′‐deoxyadenosine (F‐dAdo) to the cytotoxic drug, 2‐fluoroadenine (F‐Ade). This cytotoxicity should be restricted only to the tumor microenvironment, because the endogenously expressed wild type enzyme cannot use adenosine‐based prodrugs as substrates. To gain insight into the interaction of hDM with F‐dAdo, we have determined the crystal structures of hDM with F‐dAdo and F‐Ade. The structures reveal that despite the two mutations, the overall fold of hDM is nearly identical to the wild type enzyme. Importantly, the residues Gln201 and Asp243 introduced by the mutation form hydrogen bond contacts with F‐dAdo that result in its binding and catalysis. Comparison of substrate and product complexes suggest that the side chains of Gln201 and Asp243 as well as the purine base rotate during catalysis possibly facilitating cleavage of the glycosidic bond. The two structures suggest why hDM, unlike the wild‐type enzyme, can utilize F‐dAdo as substrate. More importantly, they provide a critical foundation for further optimization of cleavage of adenosine‐based prodrugs, such as F‐dAdo by mutants of human purine nucleoside phosphorylase.     

Cytosolic heteroglycans in photoautotrophic and in heterotrophic plant cells

In plants several ‘starch-related’ enzymes exist as plastid- and cytosol-specific isoforms and in some cases the extraplastidial isoforms represent the majority of the enzyme activity. Due to the compartmentation of the plant cells, these extraplastidial isozymes have no access to the plastidial starch granules and, therefore, their in vivo function remained enigmatic. Recently, cytosolic heteroglycans have been identified that possess a complex pattern of the monomer composition and glycosidic bonds. The glycans act both as acceptors and donors for cytosolic glucosyl transferases. In autotrophic tissues the heteroglycans are essential for the nocturnal starch-sucrose conversion. In this review we summarize the current knowledge of these glycans, their interaction with glucosyl transferases and their possible cellular functions. We include data on the heteroglycans in heterotrophic plant tissues and discuss their role in intracellular carbon fluxes that originate from externally supplied carbohydrates.     

Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae

Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high V_max and a low K_m value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl₂ and ZnCl₂ acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 °C and pH 8.5 respectively. Enzyme activity was stable at 0–40 °C and at alkaline pH.     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.