Ribosyl transfer

Nucleoside phosphorylases that catalyse the reversible cleavage of purine nucleosides to the free bases and ribose-1-phosphate are found in most cells, although a phosphorylase that will cleave adenosine has so far been identified only in bacteria. Recent studies have shown that ribo- and 2 -deoxyribofurano-syltransferase activity is associated with phosphorylase activity [19, 23., 222] and that both activities reside in one enzyme, which can be converted from one form to the other by substrate or product binding [20]. Upon crystallization of the enzyme from human erythrocytes a marked decrease in the ribosyl transfer reaction was observed [21b]. 

ADP ribosylation transfer of ADP ribose from NAD+ to a protein, adrenergic nerves responding to adrenaline. 

Figure 3.14 Structures of common nucleosides whose acid-catalysed hydrolysis has been studied. Adenosine, guanosine and cytidine are three of the four common nucleosides in RNA and their 2 -deoxy derivatives in DNA, whereas uridine is found only in RNA and 2 -deox5hh5midine in DNA. Psicofuranine is an antibiotic and is not a common constituent of nucleic acids. Inosine is a commonly used substrate in investigations of enzymic ribosyl transfer.

Nucleotide C-N bond hydrolysis and phosphorolysis at the monomer level form part of purine salvage pathways and their mechanisms have been intensively investigated for pharmacological reasons. Humans can biosynthesise purine nucleosides de novo, whereas protozoal parasites such as those causing bilharzia and Chagas disease rely on hydrolysis of preformed nucleosides from the host. Finally, a series of ribosyl transfers from NAD are important in the modification of proteins by pathogens and have been studied extensively. 

The ADP-ribosyl transfer reaction has a sulfur nucleophile, as compared with water in the non-enzymatic reaction. Given the very weak dependence on nucleophile ability of this reaction, the non-enzymatic transition state with a sulfur nucleophile would be expected to be similar to that with water. 

Only very recendy, it was discovered that a class of histone deacetylases, the simiins, is dependent on NAD. Sirtuins deacetylate proteins and concomitantly cleave NAD The reaction mechanism appears to be unique for ADP-ribosyl transfers. ... 

This meeting has shown us that ADP-ribosyl transfer catalyzed by the bacterial toxins continues to be the best understood area of ADP-ribosylation. We have seen in the presentations of Dr. Collier and Dr. Gill excellent examples of continued progress in this area. Cholera toxin and diphtheria toxin represent members of distinct families of ADP-ribosyltransferases that modify arginine and hypermodified histidine residues in their target proteins, respectively. The presentation of Dr. Ui has shown us that pertussis toxin represents yet a third type of transferase, one that modifies asparagine residues in acceptor proteins. 

Carba-Nicotinamide Adenine Dinucleotide Synthesis and Enzymological Application of a Novel Inhibitor of ADP-ribosyl Transfer... 

The results of the biosynthesis experiments suggest that NAD depletion is due to an increased rate of NAD consuming reactions. NAD can be consumed by ADP-ribosyl transfer reactions which can be inhibited by 3-aminobenzamide (10). When 5 mM 3-aminobenzamide was added to the culture medium, total inhibition of NAD depletion was observed (data not shown). Since ADP-ribosyl transfer reactions can be catalyzed by nuclear poly(ADP-ribose) polymerase and by cytoplasmic mono(ADP-ribosyl) transferases (11), the inhibition of these enzymes by 3-aminobenzamide was quantitatively compared. Fig. 2 shows dose response curves for the inhibition of purified poly(ADP-ribose) polymerase (12) and mono(ADP-ribosyl) transferase (13) by this compound. The activity of poly(ADP-ribose) polymerase was much more sensitive to inhibition with a 50% inhibitory concentration (IC50) of approximately 5.5 xM under the assay conditions used compared to an IC50 of 2000 iM for the mono(ADP-ribosyl) transferase. The effect of different concentrations of 3-aminobenzamide on NAD depletion in CF-3 cells incubated with Ca + depleted medium was also determined (Fig. 2). These cell experiments indicated an IC50 similar to that of mono(ADP-ribosyl) transferases and argue that NAD depletion in CF-3 cells was due to a stimulation of cellular mono(ADP-ribosyl)ation. 

The mono(ADP-ribosyl) transfer reactions detected here may have a G-protein as an acceptor of the ADP-ribose moiety. G-proteins have been shown to be involved in Ca + mobilization, and mono(ADP-ribosyl)ation reactions have been linked to their regulation (5-8). Therefore, the stimulation of the mono(ADP-ribosyl)ation of a G-protein, an NAD consuming reaction, could explain the loss of NAD in response to low extracellular Ca + levels in HDF. 

Uridine phosphorylase participates in ribosyl transfer reactions which take place by the following mechanism. Ribose 1-phosphate produced by phosphorolysis of one nucleoside may serve as the ribose source in the phosphoiylase-catalyzed synthesis of a different nucleoside ... 

For example, intact Ehrlich ascites tumor cells, or extracts therefrom, transfer the ribosyl group of uridine to hypoxanthine and thereby catalyze the net synthesis of inosine this reaction depends upon the coupled actions of uridine phosphorylase and purine nucleoside phosphorylase (89). Similar ribosyl transfers have been demonstrated with bacterial cells and extracts. Krenitsky has studied the kinetics of exchange between uracil-2- C and nonisotopic uridine catalyzed by highly purified uridine phosphorylase (30) ... 

Poly(ADP-ribose) polymerase is primarily a nuclear enzyme. The acceptor for the initial ADP-ribose moiety is a glutamate or the carboxyl group of a terminal lysine in the acceptor enzyme, forming an 0-glycoside. This is followed by successive ADP— ribosyl transfer to form poly(ADP-ribose), which may be a linear or branched polymer. 

Pertussis toxin is produced by the bacterium Bordetella pertussis. It covalently modifies G-proteins of the G/Go family (transfer of a ADP-ribose moiety of NAD onto G-protein a-subunits). ADP-ribosylated G-proteins are arrested in their inactive state and, as a consequence, functionally uncoupled from their respective effectors. Examples for pertussis toxin-sensitive cellular responses include the hormonal inhibition of adenylyl cyclases, stimulation ofK+ channels, inhibition of Ca2+ channels and stimulation ofthe cGMP-phosphodiesterase in retinal rods. 

Phosphoribosylpyrophosphate (PRPP) synthetase is one of the very few enzymes which transfer a pyrophosphoryl group from ATP in one step. When the synthesis is carried out in lsO-enriched water, lsO is incorporated into the PRPP, but not into AMP.91 The lsO in the PRPP arises from a pre-exchange between the H2180 and the ribose phosphate, and hence the results confirm that fission of the /5-P—O bond takes place. PRPP and ATP are starting materials in the biosynthesis of histidine, and Ai-(5 -phospho-D-ribosyl)adenosine triphosphate (29) is an intermediate. The... 

Figure 2 The actin-ADP-ribosylating toxins, (a) Molecular mode of action. The actin-ADP-ribosylating toxins covalently transfer an ADP-ribose moiety from NAD+ onto Arg177 of G-actin in the cytosol of targeted cells. Mono-ADP-ribosylated G-actin acts as a capping protein and inhibits the assembly of nonmodified actin into filaments. Thus, actin polymerization is blocked at the fast-growing ends of actin filaments (plus or barbed ends) but not at the slow growing ends (minus or pointed ends). This effect ultimately increases the critical concentration necessary for actin polymerization and tends to depolymerize F-actin. Finally, all actin within an intoxicated cell becomes trapped as ADP-ribosylated G-actin.

PolyADP-ribosylation has been reported to play a role in traumatic brain injury (TBI), excitotoxic, and oxidative injury. In the mitochondria after TBI, PARPs are activated and poIyADP-ribosylate multiple proteins involved in electron transfer. Since the ribosylation of these proteins shuts down electron transport, cells are sent into an apoptotic state. This gives insight into mitochondrial-based brain injuries and diseases. 

Histone ADP-ribosylation was first reported in 1968 [290]. Poly(ADP-ribosylation) has been implicated in several nuclear processes, including DNA replication, repair and recombination [291-294]. Histone HI and the four core histones are modified by adenosine diphospho (ADP) ribosylation which involves the transfer of the ADP-ribose moiety of NAD" " to the histone acceptor (Figs. 1 and 2). HI is the principle poly(ADP-ribosylated) histone, while core histones are ADP-ribosylated to a minor extent [295-297]. HI is modified at Glu residues 2, 14 (or 15), and 116 (or 117) and at Lys located at the C-terminus [25,298,299]. Poly(ADP-ribosylated) HI is associated with dynamically acetylated core histones [295]. There is conflicting results as to whether poly(ADP-ribosylation) of HI promotes chromatin decondensation [300-304]. 

This enzyme [EC 2.4.2.30] (also referred to as NAD+ ADP-ribosyltransferase, poly(ADP) polymerase, poly-(adenosine diphosphate ribose) polymerase, and ADP-ribosyltransferase (polymerizing)) catalyzes the reaction of NAD+ with [ADP-D-ribosyl] to produce nicotinamide and [ADP-D-ribosyl]( + i). The ADP-d-ribosyl group of NAD+ is transferred to an acceptor carboxyl group on a histone or on the enzyme itself, and further ADP-ribosyl groups are transferred to the 2 -position of the terminal adenosine moiety, building up a polymer with an average chain length of twenty to thirty units. 

Two bacterial toxins, namely pertussis toxin and cholera toxin, were of great importance in determining the function of G-proteins. Both toxins catalyze ADP ribosyla-tion of proteins. Dming ADP ribosylation, an ADP-ribose residue is transferred from NAD to an amino acid residue of a substrate protein (Fig. 5.15). 

Fig. 5.15. ADP-ribosylation of the Ga-subunit of transdudn by cholera toxin. Cholera toxin catalyzes the ADP-ribosylation of the a-subunit of the G-protein transducin. During the reaction, the ADP-ribose residue of NAD+ is transferred to Argl74 of Ga which inactivates the GTPase activity of Ga i-...

This toxin subunit is an enzyme, an ADP-ribo-syltransferase which catalyzes transfer of ADP-ribosyl units from the coenzyme NAD+ to specific arginine side chains to form N-ADP-ribosyl derivatives of various proteins. Of the proteins modified by cholera toxin, the most significant is the guanyl nucleotide regulatory protein Gs of the adenylate cyclase system.C/f/h ADP ribosylation of arginine 201 of the a subunit of protein Gs inhibits the GTP hydrolysis that normally allows the protein to relax to an unactivated form.e The ADP-ribosylated Gs keeps adenylate cyclase activated continuously and... 

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