Adenosine enzymic preparation

Selected entries from Methods in Enzymology [vol, page(s)] Adenylate kinase contamination in in phosphotransferases, 63, 7 as contaminant enzyme preparations, 64, 24 P, P -di(adenosine-5 )-tetraphosphate and P, P -di(adenosine-5 )-pentaphosphate inhibition of adenylate kinase, 63, 7, 401, 483. 

The pyrophosphorylase procedures have been applied widely for preparation of various naturally occurring a-D-glucopyranosyl esters of nucleoside pyrophosphates. For synthetic purposes, it is common to use crude or partially purified enzyme preparations, which may be a mixture of individual, specific enzymes. The synthesis of adenosine 5 -(a-D-glucopyranosyl pyrophosphate) was achieved with pyrophos-phorylases from Arthrobacter viscosus 216,217 Azotobacter vinelandii,52... 

All adenosine aminohydrolase preparations have typical absorption spectra consistent with the absence of tightly bound prosthetic groups. No dissociable cofactors are necessary for catalysis since extensive dialysis of enzyme or addition of the metal chelator ethylenediaminete-traacetate (EDTA) did not cause significant inhibition. 

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

Sulfate reduction reduction of the sulfote ion S04 " to the sulfide ion S, in which the hexavalent, positive sulfur of the sulfate is converted to the divalent, negative form. S.r. must be preceded by Sulfate activation (see). The substrate of enzymatic S.r. is therefore either adenosine phosphosulfate (APS) or phosphoadenosinephosphosulfate (PAPS). The enzy-mology of S.r. has been studied in particular in enzyme preparations from baker s yeast (Saccharomy-ces cerevisiae) and the anaerobic bacterium Desulfo-vibrio desulfuricans. The former organism performs assimilatory S.r. (see Sulfate assimilation), Ae latter dissimilatory S.r. (see Sulfate respiration). 

Nucleotidase occurs in certain leaves and plant seeds and has been purified from germinating barley. The enzyme hydrolyzes the 3 -phosphates of adenosine, inosine, guanosine, uridine, and cjrtidine as well as of coenzyme A. Purified enzyme preparations contain only traces of ATPase, acid phosphatase, and 5 -nucleotidase (126). 

These degradations generate a mixture of U, C, I, G, and A nucleosides that is directly analyzed on the ODS-2 column under the conditions described above. Inosine (I) results from the deamination of adenosine by adenosine deaminase, which is a contaminant in the enzyme preparations. If the incubation is allowed to proceed for 2 h, only U, C, I, and G can be detected. A typical analysis of total digestion of a oligonbonucleotide is shown in Fig. 3. 

The most highly purified enzyme preparation was studied for substrate specificity (Table 2). The results are expressed as per cent of nucleotide formed from each substrate as compared to adenine. Adenine phosphoribosyltransferase shows substantial activity toward 4-amino-5-imidazolecarboxamide and 2,6-diaminopurine but not hypoxanthine, guanine, 6-mercaptopurine, or adenosine. 

Catalysis by flavoenzymes has been reviewed and various analogues of FAD have been prepared e.g. P -adenosine-P -riboflavin triphosphate and flavin-nicotinamide dinucleotide ) which show little enzymic activity. The kinetic constants of the interaction between nicotinamide-4-methyl-5-acetylimidazole dinucleotide (39) and lactic dehydrogenase suggest the presence of an anionic group near the adenine residue at the coenzyme binding site of the enzyme. ... 

The bearing which these discoveries have had on the elucidation of the structure of ribopolynucleotides will be discussed later. It is important to stress here, however, that, for most purposes, the older methods of preparing nucleotides have been superseded by procedures which yield separate isomers of each. Of the techniques mentioned above, paper chromatography iB mainly of analytical value, and is the most convenient method for the qualitative detection of isomeric adenylic acids. The only disadvantage of this method is that the isomers are not completely separable from muscle adenylic acid. The presence of the latter, however, can be readily detected by hydrolyzing it to adenosine by means of the specific 5-nucleotidase present in snake venoms,66 or by deamination by a specific enzyme... 

Based on the experience mentioned previously, the Dimroth rearrangement was succesfully applied for the preparation of water-soluble macro-molecular adenosine derivatives of the redox enzymes 72 (NAD(H), NADP(H), and FAD) (Scheme IV.31) (86MI1 87MI3, 87MI2 88H1623 89MI1 90M11). 

In a number of cases there may be a contaminating enzyme present which acts on one or more of the substrates, products, or effectors of the system under study. It may be necessary to include in the reaction mixture a specific inhibitor for that contaminating activity. For example, adenylate kinase is often present in preparations of a number of phosphotransferases. It is often advantageous, in such instances, to include a specific inhibitor of adenylate kinase (e.g., P, P -di(adenosine-5 )-tetraphosphate or P P -di(adenosine-5 )-pentapho-sphate). If an inhibitor of the contaminating activity is added as an additional constituent of the reaction mixture, the investigator should demonstrate that the inhibitor is not an effector of the enzyme under study. 

Hydrolysis of sugar nucleotides with unspecific pyrophosphatases has already been mentioned (Section 11,1, p. 310). A similar reaction is catalyzed by a bacterial enzyme specific for adenosine 5 -(a-D-glucopyranosyl pyrophosphate).459 The specific conversion of uridine 5 -(a-D-glucopyranosyl pyrophosphate) into a-D-glucopyranosyl phosphate, uridine, and inorganic phosphate was observed with an enzyme from Escherichia colt 459,460 a preparation from Bacillus subtilis can act in a similar manner461 on different sugar nucleotides. ... 

Chromium(III) forms stable complexes with adenosine-S -triphosphate.840,841,842 These are kinetically inert analogues of magnesium ATP complexes and may be used to study enzyme systems. The complexes prepared are chiral and may be distinguished in terms of chirality at the metal centre (198,199).843 The related complex of chromium(lll) with adenosine-5 -(l-thiodiphosphate) has been prepared the diastereoisomers were separated.844 The stereospecific synthesis of chromium(III) complexes of thiophosphates has been reported845 by the method outlined in equation (47), enabling the configuration of the thiophosphoryl centre to be determined. The availability of optically pure substrates will enable the stereospecificity of various enzyme systems to be investigated.845... 

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