Enzymatic formation of uridine diphosphate N-acetyl-D-mannosamine.

An enzyme that catalyzes the interconversion of UDP-N-acetyl-D-glucosamine and UDP-N-acetyl-D-mannosamine was purified about 700-fold from the supernatant fraction of Bacillus cereus, and the properties of this enzyme were studied. This enzyme was not stimulated by NAD+, NADH, or any metal ions. The optimum pH was between 7.5 and 8.0. At equilibrium of the reaction, the ratio of UDP-N-acetylglucosamine to UDP-N-acetylmannosmaine was about 9:1. The enzyme was inactive toward free N-acetylhexosamines, their phosphate esters, UDP-glucose, and UDP-N-acetylgalactosamine. A stimulatory role of UDP-N-acetylglucosamine was demonstrated. In the reaction with UDP-N-acetylglucosamine, the rate as a function of substrate concentration showed a sigmoidal relationship with a Hill coefficient of 1.8 and an apparent Km value for UDP-N-acetylglucosamine of 1.1 mM. The reverse reaction with UDP-N-acetylmannosamine required the presence of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine concentration required for half-maximal activation was about 0.5 mM. The apparent Km for UDP-N-acetylmannosamine measured in the presence of 0.5 mM UDP-N-acetylglucosamine was 0.22mM. Other nucleotides or hexosamine derivatives were not stimulatory. The same activity was found in cell extracts from several bacterial species.     

Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis

Twenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2′-, 3′- and 5′-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2′- or 5′-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2′- and 5′-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3′-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2′-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.     

Cyclization of uridine monophosphate by diethyl pyrocarbonate.

Reaction between diethyl pyrocarbonate and uridine 2'-phosphate or uridine 3'-phosphate leads to the formation in high yields of uridine 2':3'-cyclic phosphate. This reaction product was identified in experiments involving (a) ultraviolet spectrophotometry, (b) paper chromatography, (c) high voltage paper electrophoresis at both pH 3.5 and 7.4, (d) acid hydrolysis, and (e) digestion with pancreatic ribonuclease.     

Studies on uridine diphosphate-galactose pyrophosphatase and uridine diphosphate-galactose: glycoprotein galactosyltransferase activities in microsomal membranes.

Rat liver microsomes showed very active uridine diphosphate-galactose pyrophosphatase activity leading to the hydrolysis of uridine diphosphate-galactose into galactose1-phosphate and finally into galactose. The activity was observed in presence of buffers with wide ranges of pH. Different concentrations of divalent cations, such as Mn²⁺, Mg²⁺, and Ca²⁺ had no significant effect on the enzyme activity. A number of nucleotides and their derivatives inhibited the pyrophosphatase activity. Of these, different concentrations of uridine monophosphate, cytidine 5′-phosphate and cytidine 5′-diphosphate have slight or no effect; cytidine 5′-triphosphate, adenosine 5′-triphosphate, guanosine 5′-triphosphate, cytidine 5′-diphosphate-glucose and guanosine 5′-diphosphate-glucose showed strong inhibitory effect whereas cytidine 5′-diphosphate-choline showed a moderate effect on the pyrophosphatase. All these nucleotides also showed variable stimulatory effects on uridine diphosphate-galactose:glycoprotein galactosyltransferase activity in the microsomes which could be partly related to their inhibitory effects on uridine diphosphate-galactose pyrophosphatase. Among them uridine monophosphate, cytidine 5′-phosphate, and cytidine 5′-diphosphate stimulated galactosyltransferase activity without showing appreciable inhibition of pyrophosphatase, cytidine 5′-diphosphate-choline, although did not inhibit pyrophosphatase as effectively as cytidine 5′-triphosphate, guanosine 5′-triphosphate, adenosine 5′-triphosphate, cytidine 5′-diphosphate-glucose, and guanosine 5′-diphosphate-glucose but stimulated galactosyltransferase activity as well as those. The fact that cytidine 5′-diphosphate-choline stimulated galactosyltransferase more effectively than cytidine 5′-phosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate suggested an additional role of the choline moiety in the system. It has been also shown that cytidine 5′-diphosphate-choline can affect the saturation of galactosyltransferase enzyme at a much lower concentration of uridine diphosphate-galactose. Most of the pyrophosphatase and galactosyltransferase activities were solubilized by deoxycholate and the membrane pellets remaining after solubilization still retained some galactosyltransferase activity which was stimulated by cytidine 5′-diphosphate-choline. In different membrane fractions a concerted effect of both uridine diphosphate-galactose pyrophosphatase and glycoprotein:galactosyltransferase enzymes on the substrate uridine diphosphate-galactose is indicated and their eventual controlling effects on the glycopolymer synthesis in vitro or in vivo need careful evaluation.     

Interaction of uridine diphosphate glucose analogs with calf liver uridine diphosphate glucose dehydrogenase. Influence of substituents at C-5 of pyrimidine nucleus.

The interaction of alpha-D-glucopyranosyl pyrophosphates of 5-X-uridines (X = CH3, NH2, CH3O, I, Br, Cl, OH) with uridine diphosphate glucose (UDPGlc) dehydrogenase (EC 1.1.1.22) from calf liver has been studied. All the derivatives investigated were able to serve as substrates for the enzyme. The apparent Michaelis constants for UDPGlc-analogs were dependent both on electronic and steric factors. Increase of substituent negative inductive effect lead to decrease of pKa for ionization of the NH-group in the uracil nucleus and, consequently, to a diminishing of the proportion of the active analog species under the conditions of assay. After correction for the ionization effect, the Km values were found to depend on the van der Waals radius of the substituent. The value of 1.95 A seems to be critical, as the analogs with bulkier substituents at C-5 showed a decreased affinity to the enzyme. The maximal velocity values of the analogs were also dependent on nature of the substituent. Good linear correlation between log V and substituent hydrophobic phi-constant was observed for a number of the analogs, although V values for the nucleotides with X = H, OH or NH2 were higher than would be expected on the basis of the correlation. The significance of the results for understanding of the topography of UDPGlc dehydrogenase active site is discussed.     

Protein engineering of uridine phosphorylase from Escherichia coli K-12. II. Comparative study of hybrid and mutant forms of uridine phosphorylases]

Genes for hybrid uridine phosphorylases (UPases) consisting of fragments of amino acid sequences of UPases from Escherichia coli and Salmonella typhimurium were constructed. Producing strains of the corresponding proteins were genetically engineered. Mutant forms of the E. coli K-12 UPase were produced by site-directed mutagenesis. A comparative study of the enzyme properties of the mutant and hybrid forms of bacterial UPases was performed. It was shown that Asp27 unlike Asp5 and Asp29 residues of the E. coli UPase forms part of the active site of the protein. A scheme of the involvement of Asp27 in the binding of inorganic phosphate is proposed.     

Problems of uridine protection

Various routes to synthesize 5'-O-dimethoxytrityl-O4-p-nitrophenylethyl- 2'-O-p-nitrophenylethylsulfonyluridine (7) as a useful intermediate in oligoribonucleotide synthesis have been investigated. The direct sulfonylation of 5'-dimethoxytrityl-O4-p-nitrophenylethyluridine (4) gave the best results despite the fact that 7 is formed in almost equal amounts with its 3'-NPES isomer (8) and the 2',3'-di-O-NPES derivative (6).     

Regulation of uridine kinase. Evidence for a regulatory site

Uridine kinase from mouse Ehrlich ascites tumor cells may exist at 4 degrees C in multiple aggregation states that only slowly equilibrate with one another. Increasing the temperature leads to dissociation, and the appearance of a single predominant species: at 22 degrees C the enzyme exists as a tetramer. There is also a break in the dependence of enzyme activity on temperature as measured in an Arrhenius plot. The feedback inhibitors CTP and UTP cause the enzyme to dissociate to the monomer, whereas the substrate ATP reverses this process. Kinetic studies show that the monomer has little or no activity. Studies of the reaction mechanism show that binding of substrates is ordered, leading to a ternary complex, and release of products is ordered: uridine is the first substrate bound, ADP the first product released. Except for the inhibitors UTP and CTP, all other nucleoside triphosphates, whether purine or pyrimidine, or containing ribose or deoxyribose, act as phosphate donor. Especially interesting are the opposite effects of CTP and dCTP on uridine kinase: unlike CTP, dCTP does not dissociate the enzyme and is competent as a phosphate donor. We propose that the various effects of different ligands are best explained by the existence of a regulatory site (with more stringent specificity than the catalytic site) that controls dissociation of uridine kinase to the inactive monomer.