AMP nucleosidase: kinetic mechanism and thermodynamics

The kinetic mechanism of AMP nucleosidase (EC 3.2.2.4; AMP + H2O----adenine + ribose 5-phosphate) from Azotobacter vinelandii is rapid-equilibrium random by initial rate studies of the forward and reverse reactions in the presence of MgATP, the allosteric activator. Inactivation-protection studies have established the binding of adenine to AMP nucleosidase in the absence of ribose 5-phosphate. Product inhibition by adenine suggests a dead-end complex of enzyme, AMP, and adenine. Methanol does not act as a nucleophile to replace H2O in the reaction, and products do not exchange into substrate during AMP hydrolysis. Thus, the reactive complex has the properties of concerted hydrolysis by an enzyme-directed water molecule rather than by formation of a covalent intermediate with ribose 5-phosphate. The Vmax in the forward reaction (AMP hydrolysis) is 300-fold greater than that in the reverse reaction. The Keq for AMP hydrolysis has been experimentally determined to be 170 M and is in reasonable agreement with Keq values of 77 and 36 M calculated from Haldane relationships. The equilibrium for enzyme-bound substrate and products strongly favors the enzyme-product ternary complex ([enzyme-adenine ribose 5-phosphate]/[enzyme-AMP] = 480). The temperature dependence of the kinetic constants gave Arrhenius plots with a distinct break between 20 and 25 degrees C. Above 25 degrees C, AMP binding demonstrates a strong entropic effect consistent with increased order in the Michaelis complex. Below 20 degrees C, binding is tighter and the entropic component is lost, indicating distinct enzyme conformations above and below 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Escherichia coli phnN,encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways

An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate --> ribose 1-phosphate --> ribose 1,5-bisphosphate --> PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + P(i) --> guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C-P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP.     

Interrelations of NAD and adenosine transformation in the rat liver

The interrelationship of NAD and adenosine (inosine) conversions in the rat liver is investigated. The ratio of products of NAD+ conversions (ADP-ribose, inosine, hypoxanthine and ribose phosphates) are established. AMP and adenosine are not detected, which indicates an availability of different activities of the corresponding enzymes. It is shown that under conditions of the high inorganic phosphate concentration (33 mM) ribose-1-phosphate, formed in the purine nucleoside phosphorylase reaction, is accumulated due to the phosphoribomutase inhibition, but in the presence of NAD+ the utilization of ribose phosphate increases significantly. Nicotinamide inhibits the NAD+-glycohydrolase reaction in the system containing 33 mM phosphate, NAD+ and adenosine and simultaneously it lowers the utilization of ribose.     

Pre-steady-state analysis of the nucleoside hydrolase of Trypanosoma vivax. Evidence for half-of-the-sites reactivity and rate-limiting product release

The nucleoside hydrolase (NH) of the Trypanosoma vivax parasite catalyzes the hydrolysis of the N-glycosidic bond in ribonucleosides according to the following reaction: beta-purine (or pyrimidine) nucleoside + H(2)O --> purine (pyrimidine) base + ribose. The reaction follows a highly dissociative nucleophilic displacement reaction mechanism with a ribosyl oxocarbenium-like transition state. This paper describes the first pre-steady-state analysis of the conversion of a number of purine nucleosides. The NH exhibits burst kinetics and behaves with half-of-the-sites reactivity. The analysis suggests that the NH of T. vivax follows a complex multistep mechanism in which a common slow step different from the chemical hydrolysis is rate limiting. Stopped-flow fluorescence binding experiments with ribose indicate that a tightly bound enzyme-ribose complex accumulates during the enzymatic hydrolysis of the common purine nucleosides. This is caused by a slow isomerization between a tight and a loose enzyme-ribose complex forming the rate-limiting step on the reaction coordinate.     

Evidence for a novel metabolic pathway of (ADP-ribose)n: pyrophosphorolysis of ADP-ribose in HeLa S3 cell nuclei

ADP-ribose liberated from (ADP-ribose)n by the action of (ADP-ribose)n glycohydrolase was converted to ATP and ribose 5-phosphate (ribose 5-P) in the presence of pyrophosphate (PPi) in HeLa S3 cell nuclei. This reaction was reversible and dependent on the simultaneous presence of ADP-ribose, PPi, Mg2+, and nuclei. These results suggest the presence of a novel enzyme in the nuclei, designated as ADP-ribose pyrophosphorylase, which catalyzes the reaction shown in Equation 1. ADP-ribose + PPi in equilibrium ATP + Ribose 5-P (1) This reaction could represent a pathway for the biosynthesis of ATP from (ADP-ribose)n in eukaryotic cell nuclei.     

Unusual binding sites for horseradish peroxidase on the surface of cultured and isolated mammalian cells. Suppression of binding by certain nucleotides and glycoproteins, and a role for calcium

Binding sites for horseradish peroxidase (HRP), with unusual properties, were detected on the surface of cultured and isolated cells after the cells (on cover slips) had been quickly dried, fixed in cold methanol, and post-fixed in a paraformaldehyde solution. The reaction for surface-bound HRP was suppressed by micromolar concentrations of glycoproteins such as invertase, equine luteinizing hormone (eLH) or human chorionic gonadotropin (hCG). The reaction was also suppressed by 20 mM CDP, UDP, GTP, NAD, and ribose 5-phosphate. Two to six times higher concentrations of GMP, fructose 1-phosphate, galactose 6-phosphate, mannose 6-phosphate, fructose 6-phosphate, and glucose 6-phosphate were required to suppress the binding reaction. AMP, ATP, heparin, mannan, and eight non-phosphorylated sugars showed relatively low competing potencies but fucoidin and alpha-lactalbumin were strong inhibitors. No addition of Ca2+ was required for the binding of HRP to the cell surface. However, calcium-depleted, inactive HRP did not compete with the binding of native (calcium-containing) HRP whereas H2O2-inactivated HRP suppressed the binding. GTP, NAD, ribose 5-phosphate, and EGTA accelerated the release of previously-bound HRP from the cell surface whereas glycoproteins (invertase, eLH, and hCG) did not do so. Addition of Ca2+ to GTP, NAD, ribose 5-phosphate or to EGTA prevented the accelerated release of HRP from the cell surface. It is suggested that calcium, present either in the surface membrane or in HRP itself, is involved in the binding of HRP to the cell surface and in the inhibition of binding by GTP, NAD, and ribose 5-phosphate. It is also suggested that alpha-lactalbumin, GTP, UDP, and CDP compete with the binding of HRP to a glycosyltransferase on the cell surface.     

Chemiluminescent oxidation of ribose catalyzed by horseradish peroxidase in presence of hydrogen peroxide

The reaction of ribose with horseradish peroxidase in the presence of H₂O₂ is accompanied by light emission. The detection of horseradish peroxidase Compound II (FeO²⁺) indicates that the enzyme participates in a normal peroxidatic cycle. Hydrogen peroxide converts horseradish peroxidase into Compound I (FeO³⁺) which in turn is converted into Compound II by abstracting a hydrogen atom from ribose forming a ribosyl radical. In aerated solutions oxygen rapidly adds to the ribosyl radical. Based on the spectral characteristics and the enhancement of the chemiluminescence by chlorophyll-a, xanthene dyes, D₂O and DABCO, it is suggested that the excited species, apparently triplet carbonyls and ¹O₂, are formed from the bimolecular decay of the peroxyl radicals via the Russell mechanism.     

Regulation of energy metabolism in macrophages during hypoxia. Roles of fructose 2,6-bisphosphate and ribose 1,5-bisphosphate

Macrophages can adapt to the absence of oxygen by switching to anaerobic glycolysis. In this study, we investigated (a) the roles of fructose 2,6-bisphosphate (Fru-2,6-P2) and ribose 1,5-bisphosphate (Rib-1,5-P2), potent activators of phosphofructokinase, (b) the enzymes responsible for the synthesis of Rib-1,5-P2, and (c) the mechanisms of regulation of these enzymes in H36.12j macrophages during the initial phase of hypoxia. Within 1 min after initiating hypoxia, glycolysis was activated through activation of phosphofructokinase. Over the same period, Fru-2,6-P2 decreased 50% and recovered completely upon reoxygenation. Similar changes in cAMP levels were observed. In contrast, the Rib-1,5-P2 concentration rapidly increased to a maximum level of 8.0 +/- 0.9 nmol/g cell 30 s after hypoxia. Thus, Rib-1,5-P2 was the major factor increasing the rate of glycolysis during the initial phase of hypoxia. Moreover, we found that Rib-1,5-P2 was synthesized by two steps: the ribose-phosphate pyrophosphokinase (5-phosphoribosyl-1-pyrophosphate synthetase; PRPP synthetase) reaction (EC ) catalyzing the reaction, Rib-5-P + ATP --> PRPP + AMP and a new enzyme, "PRPP pyrophosphatase" catalyzing the reaction, PRPP --> Rib-1,5-P2 + P(i). Both PRPP synthetase and PRPP pyrophosphatase were significantly activated 30 s after hypoxia. Pretreatment with 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine and calphostin C prevented the activation of ribose PRPP synthetase and PRPP pyrophosphatase as well as increase in Rib-1,5-P2 and activation of phosphofructokinase 30 s after hypoxia. These data suggest that the activation of the above enzymes was mediated by protein kinase C acting via activation of phosphatidylinositol specific phospholipase C in the macrophages during hypoxia.     

Deoxyribose 1-phosphate: radioenzymatic and spectrophotometric assays

A method has been developed to measure deoxyribose 1-phosphate in the presence of ribose 1-phosphate and other sugar phosphates. The specificity of the method is based on the observation that only deoxyribose 1-phosphate is hydrolyzed by heating at pH 7.4, while both deoxyribose 1-phosphate and ribose 1-phosphate remain unchanged when heated at pH 10. A tissue extract is heated at pH 10. The amount of deoxyribose 1-phosphate plus ribose 1-phosphate is determined from that of deoxyinosine plus inosine formed in a coupled enzymatic reaction, based on the following two-stage transformation: deoxyribose 1-phosphate (ribose 1-phosphate) + adenine in equilibrium deoxyadenosine (adenosine) + inorganic phosphate, catalyzed by adenosine phosphorylase; deoxyadenosine (adenosine) + H2O----deoxyinosine (inosine), catalyzed by adenosine deaminase. By taking advantage of its unique heat lability, deoxyribose 1-phosphate is eliminated by heating the tissue extract at pH 7.4, and ribose 1-phosphate is determined as above. The amount of deoxyribose 1-phosphate stems from the difference between the amount of deoxyinosine plus inosine measured in the tissue extract heated at pH 10 and that of inosine measured in the tissue extract heated at pH 7.4. Free deoxyribose 1-phosphate has been found in rat tissues, as well as in Bacillus cereus during stationary phase of growth.     

Detection of dideoxyosone intermediates of glycation using a monoclonal antibody: characterization of major epitope structures

Glycation or the Maillard reaction in proteins forms advanced glycation end products (AGEs) that contribute to age- and diabetes-associated changes in tissues. Dideoxyosones, which are formed by the long-range carbonyl shift of the Amadori product, are newly discovered intermediates in the process of AGE formation in proteins. They react with o-phenylenediamine (OPD) to produce quinoxalines. We developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate coupled to keyhole limpet hemocyanin. The antibody reacted strongly with ribose and fructose (+OPD)-modified RNase A and weakly with glucose and ascorbate (+OPD)-modified RNase A. Reaction with substituted quinoxalines indicated that this antibody favored the 2-methyl group on the quinoxaline ring. We used high performance liquid chromatography to isolate and purify three antibody-reactive products from a reaction mixture of N alpha-hippuryl-L-lysine+ribose+OPD. The two most reactive products were identified as diastereoisomers of N1-benzoylglycyl-N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and the other less reactive product as N1-benzoylglycyl-N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine. Our study confirms that dideoxyosone intermediates form during glycation and offers a new tool for the study of this important pathway in diabetes and aging.     

A coupled optical enzyme assay for phosphopentomutase

Published assays for phosphopentomutase activity are based on acid lability differences between ribose 1-phosphate and ribose 5-phosphate. The present work describes a new method in which the isomerization of ribose 5-phosphate to ribose 1-phosphate is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: ribose 1-phosphate + adenine ? phosphate + adenosine (adenosine phosphorylase); adenosine + H₂O → inosine + NH₃ (adenosine deaminase). The method has been used to show some properties of Escherichia coli phosphopentomutase.     

Proton magnetic resonance studies of 2''-,3''-, and 5''-deoxyadenosine conformations in solution.

Proton magnetic resonance studies of 2'-deoxyadenosine (2'-dA), 3'-deoxyadenosine (3'-dA), 5'-deoxyadenosine (5'-dA) and 8-bromo-5'-deoxyadenosine (8-Br-5'-dA) have been carried out in the temperature range between -60 degrees and +40 degrees C for ND3 solutios, +40 degrees and +100 degrees C for D2O solutions, and finally +10 degrees and +60 degrees C for pyridine solutions. The analysis is based on the two-state S in equilibrium N model of the ribose moiety proposed by Altona and Sundaralingam. In all solvents, 2'-dA favours slightly the S state of the ribose and the g+ conformer of the exocyclic CH2OH group. However, 3'-dA prefers strongly the N state of the ribose and the g+ conformation. Both the S and N states of the ribose are equally favoured by 5'-DA and 8-Br-5'-dA. Evidence for the existence of an intramolecular hydrogen bond between 0(5') and N3 in purine (beta)-nucleosides is presented. It is also concluded that cordycepin (3'-dA) exists in solution mainly in the anti conformation of the base relative to the ribose.     

Competition between Li+ and Mg2+ for ATP and ADP in aqueous solution: a multinuclear NMR study

We used 7Li NMR spin-lattice relaxation times and 31P NMR chemical shifts to study the binding of Li+ and Mg2+ to the phosphate moieties of ATP and ADP. To examine the binding of Li+ and Mg2+ to the base and ribose moieties, we used 1H and 13C NMR chemical shifts. The 7Li NMR relaxation times of Li+/Mg2+ mixtures of ATP or ADP increased with increasing concentrations of Mg2+, suggesting competition between the two ions for adenine nucleotides. No significant binding of Li+ and Mg2+ to the base and ribose moieties occurred. At the pH and ionic strength used, 2:1 and 1:1 species of the Li(+)-ATP and Li+-ADP complexes were present, with the 2:1 species predominating. In contrast, 1:1 species predominated for the Mg(2+)-ADP and Mg(2+)-ATP complexes. We calculated the Li(+)-nucleotide binding constants in the presence and absence of Mg2+ and found them to be somewhat greater in the presence of Mg2+. Although competition between Li+ and Mg2+ for ATP and ADP phosphate binding sites in solution is consistent with the 31P chemical shift data, the possibility that the Li+ and Mg2+ form mixed complexes with the phosphate groups of ATP or ADP cannot be ruled out.     

Activity of NMN+, nicotinamide ribose and analogs in alcohol oxidation promoted by horse-liver alcohol dehydrogenase. Improvement of this activity and structural requirements of the pyridine nucleotide part of the NAD+ coenzyme

Kinetic constants of horse-liver alcohol-dehydrogenase-mediated oxidation of alcohol by nicotinamide mononucleotide and nicotinamide ribose were determined and the role of different adenine moieties complementing the reaction mixtures was investigated. Five nicotinamide ribose analogs were synthesized and their activities as NAD+ inhibitors and as cofactors in this dehydrogenase-mediated oxidation of alcohol were assayed. In the light of these results, structural requirements of the pyridine ribofuranosyl part of the NAD+ are discussed.     

Role of the catalytic metal during polymerization by DNA polymerase lambda

The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.