Enzymes nucleic acid hydrolysis

Water is not just the solvent in which the chemical reactions of living cells occur it is very often a direct participant in those reactions. The formation of ATP from ADP and inorganic phosphate is an example of a condensation reaction in which the elements of water are eliminated (Fig. 2-22a). The reverse of this reaction— cleavage accompanied by the addition of the elements of water—is a hydrolysis reaction. Hydrolysis reactions are also responsible for the enzymatic depolymerization of proteins, carbohydrates, and nucleic acids. Hydrolysis reactions, catalyzed by enzymes called... 

Yeast extracts are concentrates of soluble material derived from yeast following hydrolysis of the cell material, particularly the proteins, carbohydrates and nucleic acids. Hydrolysis is carried out by use of the yeast s own hydrolytic enzymes (autolysis) or by other methods (hydrolysis or plasmolysis). Yeast extracts are commercially available as powders and pastes. 

A. Role of Metal Ions in Enzyme-Catalyzed Nucleic Acid Hydrolysis... 

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

The nucleic acids DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are biological polymers that act as chemical carriers of an organism s genetic information. Enzyme-catalyzed hydrolysis of nucleic acids yields nucleotides, the monomer units from which RNA and DNA are constructed. Further enzyme-catalyzed hydrolysis of the nucleotides yields nucleosides plus phosphate. Nucleosides, in turn, consist of a purine or pyrimidine base linked to Cl of an aldopentose sugar—ribose in RNA and 2-deoxyribose in DNA. The nucleotides are joined by phosphate links between the 5 phosphate of one nucleotide and the 3 hydroxyl on the sugar of another nucleotide. 

Another important group of hydrolytic enzymes are phospho- and cyclophosphodiesterases. They catalyze the hydrolysis of phospho-diester bonds and many of the most relevant biological substrates are nucleic acids. Phospholipase C and D are also important examples. Initial attempts to measure phosphodiesterase activity placed a phosphodiester between a fluorophore and a quencher and the probe was tested in vitro [146], This system was slightly modified by Caturla and used for the identification of catalysts with phosphodiesterase activity [147], More recently, Nagano and co-workers used a coumarin donor and fluorescein as a FRET... 

In the past, dissociation of the nucleoprotein complex has been brought about by salt solutions or by heat denaturation,129 but, more recently, decomposition has been effected by hydrolysis with trypsin,126 or by the use of dodecyl sodium sulfate130 or strontium nitrate.131 Some virus nucleoproteins are decomposed by ethyl alcohol.132 This effect may be similar to that of alcohol on the ribonucleoproteins of mammalian tissues. If minced liver is denatured with alcohol, and the dried tissue powder is extracted with 10% sodium chloride, the ribonucleoproteins are decomposed to give a soluble sodium ribonucleate while the deoxyribonucleoproteins are unaffected.133 On the other hand, extraction with 10 % sodium chloride is not satisfactory unless the proteins have first been denatured with alcohol. Denaturation also serves to inactivate enzymes of the tissues which might otherwise bring about degradation of the nucleic acid during extraction. 

A suitable method for this was introduced by Caspersson (mid-1920s-ca. 1940) who designed and successfully exploited UV microscopy so that the extent of the absorbtion could be determined quantitatively. Selective hydrolysis by RNAase or DNAase was used so that the DNA content of the cell could be estimated. The procedure still required reproducible preparation of sections to allow light to be transmitted and the enzymes to get access to the nucleic acids. 

RNA and DNA polymerases catalyze the same reaction mechanistically, involving hydrolysis of a nucleotide triphosphate to release pyrophosphate and form a phosphodiester bond. In both cases, the order of nucleotide addition is specified by the template, and synthesis of the growing nucleic acid chain is in a 5 to 3 direction (the enzymes move in a 3 to 5 direction along the template strand). In addition to the obvious difference in substrates (RNA polymerase utilizes ribonucleotides, whereas DNA polymerase utilizes deoxyribonucleotides), these two enzymes differ in their requirements for initiating synthesis ... 

Development of an enzymic hydrolysis by Klein32 afforded the possibility of preparing with comparative ease quantities of mixed nucleotides from desoxypentose nucleic acid, but the problem remained of obtaining the individual compounds in pure form by separation of the mixture. The development of chromatographic methods and of ion-... 

The discovery of a small proportion of a nucleoside containing thymine42 in the ribonucleic acid of two strains of Escherichia coli, in Aerobacter aero-genes, and in commercial, yeast-ribonucleic acid emphasizes the point made previously,26-28 namely, that the nucleic acids may contain constituents other than those heretofore identified. Alkaline hydrolysis of the ribonucleic acid from E. coli gave nucleotides42 (probably the 2- and 3-phosphate esters) which were converted to the nucleoside with prostatic phospho-monoesterase.62 Enzymic hydrolysis of the nucleic acid preparation also led to the nucleoside, which was degraded further to thymine by hydrolysis with perchloric acid.42 There can be little doubt that this carbohydrate derivative of thymine is intimately bound as part of the polynucleotide chain of this particular ribonucleic acid. 

In a given phosphodiester bond, hydrolytic enzymatic cleavage can occur at two locations, indicated by p and d in Figure 10.16. The former is proximal with respect to the 3 -OH group the latter is distal with respect to the 3 -OH. Enzymes that catalyze the hydrolysis of nucleic acids are nucleases (see Table 10.2). Exonucleases remove nucleotides (or nucleosides) from either the 5 or the 3 end of the polynucleotide. These are specific for either the p or the d bond. Thus, an exo-... 

The general scheme for the degradation of nucleic acids has much in common with that of proteins. Nucleotides are produced by hydrolysis of both dietary and endogenous nucleic acids. The endogenous (cellular) polynucleotides are broken down in lysosomes. DNA is not normally turned over rapidly, except after cell death and during DNA repair. RNA is turned over in much the same way as protein. The enzymes involved are the nucleases deoxyribonucleases and ribonucleases hydrolyze DNA and RNA, respectively, to oligonucleotides which can be further hydrolyzed (Fig. 15-18), so eventually purines and pyrimidines are formed. 

Hydrolytic catalysis by metal ions is also important in the hydrolysis of nucleic acids, especially RNA (36). Molecules of RNA that catalyze hydrolytic reactions, termed ribozymes, require divalent metal ions to effect hydrolysis efficiently. Thus, all ribozymes are metalloenzymes (6). There is speculation that ribozymes may have been the first enzymes to evolve (37), so the very first enzymes may have been metalloenzymes Recently, substitution of sulfur for the 3 -oxygen atom in a substrate of the tetrahymena ribozyme has been shown to give a 1000-fold reduction in rate of hydrolysis with Mg2+ but no attenuation of the hydrolysis rate with Mn2+ and Zn2+ (38). Because Mn2+ and Zn2+ have stronger affinities for sulfur than Mg2+ has, this feature provides strong evidence for a true catalytic role of the divalent cation in the hydrolytic mechanism, involving coordination of the metal to the 3 -oxygen atom. Other examples of metal-ion catalyzed hydrolysis of RNA involve lanthanide complexes, which are discussed in this volume. 

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