Enzymes Of Nucleic Acid Metabolism Research Articles

ChemInform Abstract: Biologically Important Nucleosides: Modern Trends in Biotechnology and Application

AbstractReview: 90 refs.

Biologically important nucleosides: modern trends in biotechnology and application

Modern trends in biotechnology of nucleosides of biochemical and medicinal importance are outlined. Emphasis is made on the enzymatic transformations within the general chemo-enzymatic approach to the synthesis of nucleosides. Some aspects of (i) the functioning of enzymes of nucleic acid metabolism of interest as biocatalysts, (ii) the transglycosylation reaction using sugar modified nucleosides as donors of carbohydrate residues and heterocyclic bases as acceptors catalyzed by nucleoside phosphorylases and N -deoxyribosyltransferases, (iii) the miscellaneous transformations employing D-pentofuranose 5-phosphates or α-D-pentofuranose 1-phosphates as universal glycosylatig substrates, (iv) the retrosynthesis, and (v) the one-pot transformation of D-pentoses into nucleosides are considered.

Distribution of enzymes of nucleic acid metabolism in cow snout epidermis

Certain enzymes participating in the degradation of DNA have been measured in cow snout epidermis, liver, kidney and intestinal epithelium. High activities were found in epidermis compared to other tissues. The enzyme profile of the epidermis was investigated in relation to depth. Those enzymes directly concerned with DNA degradation occurred mainly in the upper (keratinizing) layer.

Transition state analogues for enzymes of nucleic acid metabolism.

Knowledge of enzymatic transition states permits the logical design of transition state analogues. Kinetic isotope effects have been used to solve transition state structures for several N-ribosyltransferases. Nucleoside hydrolases from protozoan parasites show ribooxacarbenium ion character at their transition states, but with different extents of activation at the leaving group and the oxacarbenium ion. Transition state analogues are designed to capture these interactions and provide isozyme-specific inhibitors. Ricin A-chain is an RNA N-ribohydrolase for a single site on 28S rRNA. Its transition state resembles a fully dissociated ribooxacarbenium ion. A transition state analogue for ricin A-chain mimics the fully dissociated purine and cationic ribosyl transition state. The transition state for human purine nucleoside phosphorylase (PNP) is more dissociated than for the bovine enzyme. Immucillin-H, a powerful transition state inhibitor for human PNP, has entered clinical trials as an anti T-cell agent.

Disaccharide nucleosides and oligonucleotides on their basis. New tools for the study of enzymes of nucleic acid metabolism.

The main structural features of an important group of natural compounds, disaccharide nucleosides, are reviewed. The synthesis and properties of modified oligonucleotides on their basis as well as the methods of introduction of reactive aldehyde groups are described. The last part is devoted to the application of these compounds for studies of enzymes of nucleic acid metabolism.

The yeast mitochondrial transport proteins: new sequences and consensus residues, lack of direct relation between consensus residues and transmembrane helices, expression patterns of the transport protein genes, and protein–protein interactions with other proteins

Mitochondrial transport proteins (MTP) typically are homodimeric with a 30-kDa subunit with six transmembrane helices. The subunit possesses a sequence motif highly similar to Pro X Asp/Glu X X Lys/Arg X Arg within each of its three similar 10-kDa segments. Four (YNL083W, YFR045W, YPR021C, YDR470C) of the 35 yeast (S. cerevisiae) MTP genes were resequenced since the masses of their proteins deviate significantly from the typical 30 kDa. We now find these four proteins to have 545, 285, 902, and 502 residues, respectively. Together with only four other MTPs, the sequences of YPR021C and YDR470C show substitutions of some of the five residues that are absolutely conserved among the 12 MTPs with identified transport function and 17 other MTPs. We do now find these five consensus residues also in the new sequences of YNL083W and YFR045W. Additional analyses of the 35 yeast MTPs show that the location of transmembrane helix sequences do not correlate with the general consensus residues of the MTP family; protein segments connecting the six transmembrane helices and facing the intermembrane space are not uniformly short (about 20 residues) or long (about 40 residues) when facing the matrix; most MTPs have at least one transmembrane helix for which the sum of the negative hydropathy values of all residues yields a very small negative value, suggesting a membrane location bordering polar faces of other transmembrane helices or a non-transmembrane location. The extra residues of the three large MTPs are hydrophilic and at the N-terminal. The 200-residue N-terminal segment of YNL083W has four putative Ca 2+-binding sites. The 500-residue N-terminal segment of YPR021C shows sequence similarity to enzymes of nucleic acid metabolism. cDNA microarray data show that YNL083W is expressed solely during sporulation, while the expressions of YFR045W, YPR021C, and YDR470C are induced by various stress situations. These results also show that the 35 MTP genes are expressed under a rather diverse set of metabolic conditions that may help identify the function of the proteins. Interestingly, yeast two-hybrid screens, that will also be useful in identifying the function of MTPs, indicate that MIR1, AAC3, YOR100C, and YPR011C do interact with non-MTPs.

Antiviral Nucleoside Analogues with Axial Chirality

Chemistry and biological activity of nucleoside analogues comprising a chiral axis will be reviewed with emphasis on anti-HIV agent adenallene (1a) and anti-HIV/HBV analogue cytallene (1b). Possible mechanism of action, activity toward the enzymes of nucleic acid metabolism and structure-activity relationships will be discussed.

Should the asymmetry of enzymatic active centers always correlate with the asymmetry of their substrates?

Analysis of the substrate specificity of DNA polymerases and in part some other enzymes of nucleic acid metabolism towards unnatural L-stereomeric nucleosides and nucleotides was made. As a result, the hypothesis is proposed that when the chiral part of the substrate molecule does not interact specifically with the enzyme, substitution of natural D enantiomers by L compounds is possible.

Possibility for the Existence of a General Conformational Motif in the Active Sites of Enzymes Which are Involved in Nucleic Acids Metabolism

Abstract Many different modified nucleosides and nucleotides with conformationally restricted partly flattened sugar residues are analyzed as substrates or inhibitors of several groups of enzymes of nucleic acid metabolism. A detailed examination of the sugar moiety of large group of modified nucleosides showed that there is a striking conformational similarity, i.e., they are flattened. We propose herein a hypothesis which can represent a general conformational elements in the structure of the active sites of several different groups of enzymes. This proposal envisions that during the enzymatic process natural substrates should reflect these flattened conformations. This hypothesis allows computation of conformational analyses of the enzyme actives centers as well as the design of new actively metabolized modified nucleosides.

Determination of plasma cytidine deaminase activity by HPLC.

Cytidine deaminase is an enzyme of nucleic acid metabolism, the measurement of which has been proposed as a useful test for the early detection of pre-eclamptic toxaemia in pregnancy. The enzyme converts the nucleoside cytidine to uridine, with the release of ammonia, and it is the measurement of this latter compound that forms the basis of the conventional methods for the assay of cytidine deaminase. The low activity of the enzyme requires long incubation times, which in turn increase the possibility of contamination by exogenous ammonia. We have developed a new method for determining cytidine deaminase activity, utilising high performance liquid chromatography to measure the production of uridine. This method uses much shorter incubation times and is unaffected by ammonia contamination. This paper describes the development of the method and its comparison with the established assay. The relative merits of each are discussed. Finally, the adaptation of incubation and chromatographic conditions, in order to measure other enzymes of nucleic acid metabolism which are of clinical interest, is briefly mentioned.

Enzymes of nucleic acid metabolism as biochemical markers of T cell development in mouse

The levels of three purine salvage enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and 5′-nucleotidase (5′-N), which are known to be associated with certain immunodeficiency disorders were determined in mouse T lymphocytes. These enzymes showed characteristic changes depending on the stage of T cell development. The activity of ADA was 5-fold higher in thymocytes compared to splenic T cells. On the other hand, the splenic T cells displayed a 2-fold and a 4–8 fold greater activity of PNP and 5′-N, respectively than those of thymocytes. The apparent K m and V max values have been determined for all the 3 enzymes in the immature and mature T cells. The data demonstrate that the absolute and relative activity of these enzymes may be used as biochemical markers to characterize the T lymphocytes during different stages of differentiation.

Antibiotics from basidiomycetes. XVII. The effect of marasmic acid on nucleic acid metabolism.

From submerged cultures of Lachnella villosa, Lachnella sp. 541, and Peniophora laeta we isolated marasmic acid (1), a metabolite first described from surface cultures of Marasmius conigenus. The sesquiterpenoid exhibits potent antimicrobial and cytotoxic properties. In cells of the ascitic form of Ehrlich carcinoma RNA and DNA syntheses are preferentially inhibited. Marasmic acid inhibits RNA synthesis in isolated nuclei, but does not interfere with the transport of nucleoside precursors into the cells. RNA polymerase II and capping enzyme (mRNA guanylyltransferase), two enzymes of nucleic acid metabolism, are markedly affected after preincubation with marasmic acid. We assume that marasmic acid acts on nucleic acid syntheses by direct inhibition of some of the enzymes involved. This mode of action would also explain its mutagenic properties. The preparation and testing of two derivatives, 2 and 3, revealed that the alpha,beta-unsaturated aldehyde is essential for the antimicrobial and cytotoxic activity of marasmic acid.

37] DNA-dependent RNA polymerase

An essential lysine residue in the active center of the catalytic subunit of DNA-dependent RNA polymerase can be affinity labeled with methylthioinosinedicarboxaldehyde (MMPR-OP). The c-amino group of the lysine forms a Schiff base with an aldehyde group of MMPR-OP, which can be converted to the stable amine by mild reduction with sodium borohydride. This chapter details the synthesis of unlabeled MMPR-OP, synthesis of [ 35 S]MMPR-OP. It also discusses affinity labeling DNA-dependent RNA polymerase, enzyme purification, and reduction of the Schiff base to the stable amine. The chapter concludes that in all studies carried out to date on enzymes of nucleic acid metabolism, MMPR-OP has shown specificity for lysine residues. Inhibition of other enzymes of nucleic acid metabolism by MMPR-OP can be taken as presumptive evidence for the presence of lysines in their active centers, and the procedures described above could be modified for individual study.

An alternative approach to the study of new enzymatic reactions involving DNA (DNA-dependent ATPases-purification-properties-E. coli).

Ordinarily, enzymes that catalyze bimolecular reactions involving DNA and nucleoside triphosphates are assayed in terms of the predicted changes in DNA. However, by assay of the enzymes by changes in nucleoside triphosphates, new enzymes may be sought without prior knowledge of the role of DNA in the reaction. This approach was used to isolate two new enzymes from Escherichia coli and one from T4 phage-infected E. coli. In this communication, the general application of this technique and its specific use in the isolation of two DNA-dependent ATPases from E. coli are described. Both enzymes catalyze a DNA-dependent cleavage of ATP to ADP and Pi. The two enzymes can be distinguished by their characteristic activities with different DNAs and synthetic polydeoxynucleotides. Neither of the enzymes shows endonuclease or exonuclease activity in conventional assays. These reactions were studied by isotope-exchange experiments. The discovery of these enzymes attests to the effectiveness of this method to seek new reactions involving nucleic acids. Moreover, our results suggest that other enzymes of nucleic acid metabolism may be found and purified by this new approach.

Activity of some enzymes of nucleic acid metabolism in developing rat liver.

The increase in DNA, RNA, and nitrogen as well as the activity of some enzymes involved in nucleic acid metabolism were measured during liver development. During the perinatal period (day 18 of gestation until birth) high activities of thymidine kinase and DNA polymerase were found which decline in the early suckling period, whereas the activities of the DNA-dependent RNA polymerases show a slow continuous increase. During the juvenile period (starting about 4 weeks after birth) the values of all enzymes studied approximate to those measured in liver tissue of adult animals. The correlation of these enzymes with the growth kinetics of liver tissue is discussed.

Recognition sites around the catalytic centre of DNA-dependent RNA polymerase.

WE have been studying the structure of enzymes of nucleic acid metabolism by amino-acid sequence analysis and affinity labelling techniques and hope in this way to discover specific enzyme inhibitors. We report here the inhibition of purified DNA-dependent RNA polyrnerase from Escherichia coli by the nucleoside analogues, β-D-ribosyl-6-methyl-thiopurine (MMPR, methylthioinosine) and its periodate oxidation product (MMPR-OP), and by β-D-arabinosyl-6-mercaptopurine (Ara-6MP). We also report a lack of inhibition by the arabinosides of cytosine (Ara-C) and adenine (Ara-A). The results lead us to propose a tentative stochastic model for the catalytic centre of DNA-dependent RNA polyinerase from E. coli B.

Enzymes of nucleic acid metabolism.

INTRODUCTION INTERCONVERSIONS OF PYRIMIDINE NUCLEOTIDES . Phosphorylation of nucleotides . Biosynthesis of cytidine triphosphate . Reduction of ribonucleotides . Biosynthesis of thymidylate . PYRIMIDINE NUCLEOTIDE METABOLISM AFTER T-EvEN PHAGE INFECTION . . . . Biosynthesis of deo�y-5-hydro�ymethylcytidine triphosphate . CytosVne versus hydro�ymethylcytosine ., . . , .. . . . . . , ... . . . . . . . , . . . . . . . . . Biosynthesis of thymidine triphosphate . Nonessential pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reduction of ribonucleotides . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . DEGRADATION OF HOST DNA AFTER T-EvEN PHAGE INFECTION . ..... .. . .. . . Intermediates in the degradation of host DNA . Role of bacterial nucleases . Role of phage-induced nucleases .' . . . . . . . . . . . . . . . .

Enzymes of Nucleic Acid Metabolism from Wheat Seedlings: I. Purification and General Properties of Associated Deoxyribonuclease, Ribonuclease, and 3′-Nucleotidase Activities

A nuclease has been isolated from germinating wheat seedlings and purified over 830-fold. The purified preparation hydrolyzes denatured DNA, rRNA, and the 3′-phosphoester linkage of 3′-AMP at similar rates. The preparation exhibits no appreciable 5′-nucleotidase, phosphodiesterase, or phosphomonoesterase activity. The three enzyme activities have remained associated throughout a variety of purification procedures including chromatography on several ion-exchange resins, gel filtration, and polyacrylamide disc electrophoresis at pH 9.5 and 8.3. The ratios of the three activities remain essentially constant throughout all of these procedures. The three enzyme activities exhibit a great degree of similarity with respect to the following properties: (a) pH optima, (b) requirement for Zn++ and sulfhydryl compounds at pH 4.5, (c) rate of destruction of activity by sulfhydryl compounds at pH 8, (d) stability to storage, (e) effects of temperature and duration of heating, (f) reactivation on heating between 65° and 70°, (g) effect of N-bromosuccinimide, (h) effect of metal cations, and (i) effect of inhibitors and EDTA. It is concluded that the DNase, RNase, and 3′-nucleotidase activities in all probability reside in a single protein.

Enzymes of Nucleic Acid Metabolism from Mung Bean Sprouts: II. THE SPECIFICITY OF 3‘-NUCLEOTIDASE ACTIVITY AND GENERAL PROPERTIES OF THE 3‘-NUCLEOTIDASE-RIBONUCLEASE M2 FRACTION

Abstract The 3'-nucleotidase present in mung bean sprouts was found to hydrolyze the 3'-monophosphates of adenosine, guanosine, uridine, and cytidine in the order 3'-AMP g 3'-GMP g 3'-UMP g 3'-CMP, and also to hydrolyze the 3'-phosphate group of coenzyme A, but showed no significant activity for 2'- or 5'-ribonucleotides or for several non-nucleotide phosphomonoesters. pH optima and the respective Km values for 3'-AMP, 3'-GMP, 3'-CMP, and 3'-UMP were determined. The 3'-nucleotidase and ribonuclease M2 were inactivated reversibly at pH 5 and by dialysis and irreversibly by ethylenediaminetetraacetate. Inactivation of both enzymes at pH 5 could be largely prevented by the presence of Zn++, while several other metal ions were ineffective. The optimal concentrations of Zn++ for preventing inactivation were considerably different for the 3'-nucleotidase and RNase M2. The two enzyme activities were similarly inactivated by heat at pH 5 and 7.5. The pH optimum of RNase M2 was pH 5 with no Zn++ present, but dropped to pH 4.5 in the presence of 5 x 10-5 m Zn++, which also greatly increased its activity in the pH range 3.5 to 4.5. Several lines of evidence suggesting that the 3'-nucleotidase and RNase M2 activities may be due to the same protein are presented.