Studies on transfer ribonucleic acids and related compounds. VIII(1). Further studies on aromatic phosphoramidates as a protecting group for phosphomonoesters

Stability of aromatic phosphoramidates was studied using 2',3'-O-dibenzoyluridine 5'-phosphoramidates and N,2',3'-O-tribenzoylcytidine 5'-phosphate. The effect of dicyclohexylcarbodiimide in this mixture was investigated. Decomposition of the anilidate was slower in the presence of DCC.Substituted anilidates of uridine 5'-phosphate were synthesized and the stability of these amidates in anhydrous pyridine was studied.2'-O-Benzoyluridine 3'-phosphoranilidate and the corresponding beta-naphthylidate were compared in their stabilities in anhydrous pyridine, 50% aqueous pyridine and 80% acetic acid. 2'-O-Benzoyluridine 3'-phosphoro-beta-naphthylidate was used for synthesis of dinucleotides.     

Interaction of "aza" and "deaza" analogs of adenosine cyclic 3', 5'-phosphate with some enzymes of adenosine cyclic 3', 5'-phosphate metabolism: evidence that the lone pair electrons of N-3 are involved in the binding of adenosine cyclic 3', 5'-phosphate to type II adenosine cyclic 3', 5'-phosphate-dependent protein kinase.

Five hetercyclic analogs of adenosine cyclic 3',5'-phosphate (cyclic AMP) were examined for their ability (1) to stimulate type II cyclic AMP-dependent kinases from bovine brain, bovine heart, and rat liver; (2) to serve as substrates for "high Km" (Km for cyclic AMP = 0.13-0.43 mM) cyclic nucleotide phosphodiesterases from bovine heart, rabbit kidney, and rat liver; and (3) to inhibit the hydrolysis of cyclic AMP catalyzed by "low Km" (Km for cAMP = 0.32-1.5 muM) cyclic nucleotide phosphodiesterases from bovine brain, bovine heart, dog heart, rabbit liver, rat brain and rat liver. The analogs all had a purine ring system which had been modified by replacement of a ring carbon with nitrogen or vice versa to yield 2-aza-cAMP (7-amino-4-beta-D-ribofuranosylimidazo [4,5-d] -v-triazine cyclic 3',5'-phosphate); 8-aza-cAMP (7-amino-3-beta-D-ribofuranosyl-v-triazolo-[4,5-d]-pyrimidine cyclic 3',5'-phosphate); 1 deaza-cAMP (7-amino-3-beta-D-ribofuranosylimidazo [4,5-b[pyridine cyclic 3',5'-phosphate); 3-deaza-cAMP (4-amino-1-beta-D-ribofuranosylimidazo[4,5-c]pyridine cyclic 3',5'-phosphate) and 7-deaza-cAMP (7-amino-4-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine cyclic 3',5'-phosphate).     

Synthesis and characterization of pyridine N-oxide complexes of manganese, copper and zinc

A series of metal carboxylates containing pyridine N-oxide are prepared via one pot synthesis and solid phase synthesis. The structural variations from metal to metal are observed. In the case of reactions of manganese(II) acetate with pyridine N-oxide in the presence of aromatic carboxylic acids, polymeric complexes with bridging aromatic carboxylate as well as bridging pyridine N-oxide are observed. Whereas, the reaction of copper(II) acetate with pyridine N-oxide in the presence of an aromatic carboxylic acid led to mononuclear or binuclear paddle wheel carboxylate complexes with monodentate pyridine N-oxide. Co-crystal of two neutral complexes having composition [Cu₂(OBz)₄(MeOH)₂][Cu₂(OBz)₄(pyO)₂] (where OBz = benzoate, pyO = pyridine N-oxide) each neutral parts have paddle wheel structure. Solid phase reaction of zinc chloride with sodium benzoate prepared in situ and pyridine N-oxide leads to a tetra-nuclear zinc complex.     

Immobilisation of a 5'-Phosphodiesterase from Vegetal Origin by Covalent Binding on Activated Celite

A vegetal enzyme, 5'-phosphodiesterase, has been immobilised by covalent coupling onto activated Celite. This nonmicrobial activity was obtained from barley rootlets, an economical brewer's by-product. 5'-ribonucleotides are selectively cleaved from RNA by a partially purified preparation of the enzyme. These compounds are high added value products used as flavor enhancers in food industries (disodium 5'-inosinate, disodium 5'-guanylate) and have important applications in the pharmaceutical industry (cytidine 5'-phosphate, uridine 5'-phosphate and adenine 5'-phosphate). Glutaraldehyde was used as coupling agent. A concentration of 5 mM of glutaraldehyde was found adequate. Using a charge of 160.8 units μ g carrier_-1 a total of 15% of the activity could be recovered in the carrier. The characterisation of the solid biocatalist is reported.     

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.     

The degradation of ribonucleic acid in the cotyledons of Pisum arvense

1. A ribonuclease has been partially purified from the cotyledons of germinating seed of Pisum arvense. 2. The enzyme degrades ribopolynucleotides to adenosine 3'-phosphate, guanosine 3'-phosphate and the cyclic nucleotides cytidine 2',3'-phosphate and uridine 2',3'-phosphate; no resistant ;core' remains. 3. The activity of RNA-degrading enzymes in the cotyledons increases to a maximum during the first 5 days of germination, passes through a minimum around the eighth day, and thereafter increases again. 4. Ion-exchange chromatography of methanol-soluble extracts of cotyledons revealed the presence, amongst other components, of the 2'-, 3'- and 5'-phosphates of cytidine and uridine, the 3'- and 5'-phosphates of adenosine, and guanosine 5'-phosphate. 5. Seed soaked in a solution containing [(32)P]orthophosphate gave a methanol-soluble fraction containing labelled nucleoside 5'-phosphates, but nucleoside 2'- and 3'-phosphates were not labelled. 6. It is believed that the nucleoside 2'- and 3'-phosphates arise by the action of ribonuclease on cotyledon RNA.     

Determination of pyrimidine phosphoribosyltransferase and uridine kinase activities by an assay with DEAE-paper discs

Rapid (6 min washing step) and reliable methods for determining uridine kinase and pyrimidine phosphoribosyltransferase activities were devised. These procedures, consisting of a spotting technique on DEAE-discs followed by washing and elution, permitted the consistent recovery of about 90% of the nucleoside 5'-phosphate esters formed from radioactive precursors, either uridine or 5-fluorouracil, respectively. Of these precursors, less than 2% were retained on the discs. Direct counting of the discs (without elution), filtration by either gravity or suction, and the use of so-called activation of discs have not proved advantageous.     

The pyrimidine nucleotide reductase step in riboflavin and F(420) biosynthesis in archaea proceeds by the eukaryotic route to riboflavin

The Methanococcus jannaschii gene MJ0671 was cloned and overexpressed in Escherichia coli, and its gene product was tested for its ability to catalyze the pyridine nucleotide-dependent reduction of either 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (compound 3) to 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate (compound 4) or 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (compound 7) to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (compound 5). Only compound 3 was found to serve as a substrate for the enzyme. NADPH and NADH functioned equally well as the reductants. This specificity for the reduction of compound 3 was also confirmed by using cell extracts of M. jannaschii and Methanosarcina thermophila. Thus, this step in riboflavin biosynthesis in these archaea is the same as that found in yeasts. The absence of the other genes in the biosynthesis of riboflavin in Archaea is discussed.     

Location of protein S1 of Escherichia coli ribosomes at the ''A''-site of the codon binding site. Affinity labeling studies with a 3''-modified A-U-G analog.

An affinity analog with a 5-bromoacetamido uridine 5'-phosphate moiety bonded to the 3' end of A-U-G has been prepared with the aid of polynucleotide phosphorylase. This 3'-modified, chemically reactive A-U-G analog was used to probe the ribosomal codon binding site. The yield of the reaction depended strongly on the ribosomal source and was sensitive to salt-washing ribosomes. The major crosslinking product was identified to be protein S1. Since the reaction of this 3'-modified A-U-G programmed ribosomes for Met-tRNA-Met-M binding, it is concluded that protein S1 is located at or near the 3'-side of the ribosomal codon binding site.     

Aminophospholipid glycation and its inhibitor screening system: a new role of pyridoxal 5'-phosphate as the inhibitor

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5'-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5'-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5'-phosphate, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.     

Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin-inhibited DNA synthesis

Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.     

Pyridoxamine-5'-phosphate oxidase exhibits no specificity in prochiral hydrogen abstraction from substrate

The stereochemistry for hydrogen removal from pyridoxamine 5'-phosphate with liver pyridoxine (pyridoxamine)-5'-phosphate oxidase was examined to determine whether or not there are significant steric constraints at the substrate region of the active site of the oxidase. For this, pyridoxal 5'-phosphate was reduced with tritium-labeled sodium borohydride in ammoniacal solution to yield racemically labeled [4',4'-3H]pyridoxamine 5'-phosphate which was then chemically or enzymatically oxidized to [4'-3H]pyridoxal 5'-phosphate. This latter was used as coenzyme with either L-aspartate (L-glutamate) aminotransferase and L-glutamate or L-glutamate decarboxylase and alpha-methyl-DL-glutamate to generate [4'-3H]pyridoxamine 5'-phosphate known to be labeled in the R-position. Reaction of the oxidase with the pro-R as well as the pro-R,S-labeled substrates followed by isolation of [4'-3H]pyridoxal 5'-phosphate and 3H2O revealed only half the radioactivity was abstracted from the original substrate in either case. Hence, the oxidase is not stereospecific and equally well catalyzes removal of either pro-R or pro-S hydrogen from the 4-methylene of pyridoxamine 5'-phosphate.     

Studies on transfer ribonucleic acids and related compounds. IX. Ribooligonucleotide synthesis using a photosensitive o-nitrobenzyl protection at the 2'-hydroxyl group

o-Nitrobenzyl group was introduced to the 2′-hydroxyl function of uridine via 2′,3′-O-(dibutylstannylene) uridine. The benzylated uridine was protected at the 5′-hydroxyl group with monomethoxytrityl chloride and condensed with 2′,3′-O-dibenzoyluridine 5′-phosphate or N,N′,2′,3′-O-tetrabenzoyladenosine 5′-phosphate using dicyclohexylcarbodiimide (DCC). o-Nitrobenzyl ether linkage of the dinucleotides was removed by UV irradiation with wavelength longer than 320 nm. Deprotected UpU and UpA thus obtained were characterized by RNase A digestion.     

Kinetic studies on degradation of nucleotides catalyzed by phosphodiesterase-phosphomonoesterase from Fusarium moniliforme

Degradation of the 2'-phosphates, 3'-phosphates, 5'-phosphates, 2':3'-cyclic phosphates, 3':5'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) of adenosine, guanosine, cytidine, and uridine catalyzed by Fusarium phosphodiesterase-phosphomonoesterase was followed by means of high performance liquid chromatography. All the nucleotides were susceptible to the enzyme to a greater or lesser degree, and the kinetic constants, Km and kcat, were determined at pH 5.3 and 37 degrees C. These constants were affected by both the nucleoside moiety and the position of the phosphate. Judged from kcat/Km, the 3'-phosphates, 2':3'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) were good substrates, whereas the 2'-phosphates, 5'-phosphates, and 3':5'-cyclic phosphates were poor substrates except for adenosine 2'-phosphate, adenosine 5'-phosphate, and cytidine 5'-phosphate, which were hydrolyzed relatively easily. Among the phosphodiesters, the 2':3'-cyclic phosphates of adenosine, guanosine, and cytidine; and the 3':5'-cyclic phosphates of adenosine and cytidine were degraded into nucleoside and inorganic phosphate without release of intermediary phosphomonoester into the medium. Other phosphodiesters were degraded stepwise releasing definite intermediates.     

Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5''-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate.     

Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors

The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with 31P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH2 group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1.(ABSTRACT TRUNCATED AT 250 WORDS)     

An enzymatic method for the synthesis of [14C]pyridoxal 5'-phosphate from [14C]pyridoxine

A new enzymatic method for the synthesis of [14C]pyridoxal 5'-phosphate is presented. [14C]Pyridoxal 5'-phosphate was synthesized from [14C]pyridoxine through the successive actions of pyridoxal kinase and pyridoxamine 5'-phosphate oxidase in a reaction mixture containing ATP, [14C]pyridoxine, and both enzymes. [14C]Pyridoxal 5'-phosphate was isolated by omega-aminohexyl-Sepharose 6B column chromatography. The overall yield of the product was more than 60%, starting from 550 nmol of [14C]pyridoxine. The radiochemical purity of the products, as determined by thin-layer and ion-exchange chromatography, was greater than 98%.     

His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis.

The three-dimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3' of pyridoxal-5'-phosphate and other residues at the active site such as S203, K257, H357 and R402 (numbering as per the human enzyme). This and the conserved nature of H231 in all serine hydroxymethyltransferases highlights its importance in catalysis and/or maintenance of oligomeric structure of the enzyme. In an attempt to decipher the role of H230 (H231 of the human enzyme) in the catalytic mechanism and/or maintenance of oligomeric structure of sheep liver serine hydroxymethyltransferase, the residue was mutated to arginine, phenylalanine, alanine, asparagine or tyrosine. Our results suggest that the nature of the amino acid substitution has a marked effect on the catalytic activity of the enzyme. H230R and H230F mutant proteins were completely inactive, dimeric and did not bind pyridoxal-5'-phosphate. On the other hand, mutation to alanine and asparagine retained the oligomeric structure and ability to bind pyridoxal-5'-phosphate. These mutants had only 2-3% catalytic activity. The side reactions like transamination and 5,6,7, 8-tetrahydrofolate independent aldol cleavage were much more severely affected. They were able to form the external aldimine with glycine and serine but the quinonoid intermediate was not observed upon the addition of 5,6,7,8-tetrahydrofolate. Mutation to tyrosine did not affect the oligomeric structure and pyridoxal-5'-phosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The kcat / Km values for L-serine and Lallothreonine were 10-fold and 174-fold less for this mutant enzyme compared to the wild-type protein. These results suggest that H230 is involved in the step prior to the formation of the quinonoid intermediate, possibly in orienting the pyridine ring of the cofactor, in order to facilitate effective proton abstraction.     

Intracellular predominance of the pyridoxal 5'-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances

The intracellular proportion of the pyridoxal 5'-phosphate form of aspartate aminotransferase to the total enzyme in E. coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5'-phosphate form of the enzyme by extracellularly added sodium borohydride. A large portion (80-99%) of the intracellular aspartate aminotransferase was in pyridoxal 5'-phosphate form in both natural and synthetic medium-grown bacterial cells. The intracellular predominancy of pyridoxal 5'-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values. In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5'-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5'-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract. Various extracellularly-added vitamin B6 antagonists decreased the intracellular amount of pyridoxal 5'-phosphate without decrease in the total intracellular activity of the enzyme. The modified forms were stable in E. coli B cells and reversed into pyridoxal 5'-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal. The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5'-phosphate and apoaspartate aminotransferase. The fact that about 50% of the intracellular pyridoxal 5'-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified aspartate aminotransferase was reversed to pyridoxal 5'-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5'-phosphate forms of aspartate aminotransferase in E. coli cells.     

Structure and mechanism of Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of either pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP), forming pyridoxal 5'-phosphate (PLP). This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli and as a part of the salvage pathway of this coenzyme in both E. coli and mammalian cells. Recent studies have shown that in addition to the active site, PNPOx contains a noncatalytic site that binds PLP tightly. The crystal structures of PNPOx with one and two molecules of PLP bound have been determined. In the active site, the PLP pyridine ring is stacked almost parallel against the re-face of the middle ring of flavin mononucleotide (FMN). A large protein conformational change occurs upon binding of PLP. When the protein is soaked with excess PLP an additional molecule of this cofactor is bound about 11 A from the active site. A possible tunnel exists between the two sites. Site mutants were made of all residues at the active site that make interactions with the substrate. Stereospecificity studies showed that the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon of PMP. The crystal structure and the stereospecificity studies suggest that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.