Purine nucleosides, enzymic action

The susceptibilities of some of these fluorinated purine nucleosides to the action of enzymes are now described. In contrast to the inertness of the 2 -deoxy-2 -fluoro- and 3 -deoxy-3 -fluorocytidine analogs 739, 744, and 821 towards cytidine deaminase, the adenosine compounds 867, 883, and 906 are readily deaminated - by the adenosine deaminase in erythrocytes and calf intestine, but the resulting (deaminated) inosine compounds (from 867 and 883), as well as 888, are highly resistant - to cleavage by purine nucleoside phosphorylase (to give hypoxanthine base for the first two). The reason was discussed. Both 867 and 883 can form the 5 -triphosphates, without deamination, in human erythrocytes or murine sarcoma cells in the presence of 2 -deoxycoformycin, an adenosine deaminase inhibitor, but... 

A close look at this reaction reveals that in the opposite direction, the reaction is of the phosphorolysis type. For this reason, the enzymes catalyzing the reaction with ribose-l-phosphate are called phosphorylases, and they also participate in nucleic acid degradation pathways. Purine nucleoside phosphorylases thus convert hypoxanthine and guanine to either inosine and guanosine if ribose-l-phosphate is the substrate or to deoxyinosine and deoxyguanosine if deoxyribose-1-phosphate is the substrate. Uridine phosphorylase converts uracil to uridine in the presence of ribose-l-phosphate, and thymidine is formed from thymine and deoxyribose-l-phosphate through the action of thymidine phosphorylase. 

Purine nucleosides are cleaved by the action of purine nucleoside phosphorylase with the liberation of ribose 1-phosphate (Kl, PI). The enzyme is apparently specific for purines. The material from erythrocytes catalyzes the phosphorolysis of purine but not pyrimidine nucleosides (T6.) Purine phosphorylase activity is found widespread in nature and in many animal tissues (FIO). Friedkin and Kalckar investigated an enzyme capable of cleaving purine deoxynucleosides to the aglycone and deoxy-ribose 1-phosphate. They concluded that the enzyme was identical to that which splits purine ribonucleosides (F8, F9). This enzyme is capable of degrading inosine, xanthosine, and guanosine to forms readily attacked by other enzymes. In so doing, it permits living cells to retain the ribose and deoxyribose moieties. 

Purine-pyrimidine deoxyribosyl transfer reactions result when reactions (1) and (2) are catalyzed by the joint actions of purine nucleoside phosphorylase and thymidine phosphorylase, that is, when the activities of these enzymes are coupled. For example, the following reaction is catalyzed by extracts of human leukocytes (18) ... 

For the past decade this laboratory has devoted much of its attention to an examination of various facets of purine metabolism in human erythrocytes. These cells do not have the complete pathway for the novo synthesis of purines and do not make nucleic acids. On the other hand, they have an active nucleotide metabolism and contain the salvage enzymes, hypoxan-thine-guanine phosphoribosyl transferase (HGPRTase), adenine phosphoribosyl transferase (APRTase) and adenosine kinase. In view of the fact that the activities of certain enzymes of purine metabolism are quite high (e.g., purine nucleoside phos-phorylase occurs at a level of about 15 umolar units/ml of erythrocytes) and the total mass of erythrocytes in the adult human being is in excess of two liters, it appears that these cells play an important and perhaps not yet fully appreciated role in the whole body economy of purines in man. Therefore, we believe that the human erythrocyte provides a very useful model system for the examination of purine metabolism in man as well as for investigations of the action of certain purine and purine nucleoside antimetabolites, many of which are important in medicine. 

The purine and pyrimidine bases can be converted to then-respective nncleotides by reaction with 5-phosphoribosyl 1-pyrophosphate. Since these bases are not very soluble, they are not transported in the blood, so that the reactions are only of qnantitative significance in the intestine, where the bases are produced by degradation of nucleotides. In contrast, in some cells, nucleosides are converted back to nucleotides by the activity of kinase enzymes. In particular, adenosine is converted to AMP, by the action of adenosine kinase, and uridine is converted to UMP by a uridine kinase... 

Antiviral drugs are often antimetabolites that are structural analogs of purine or pyrimidine bases or j their nucleoside forms.. Many are pro-drugs to be activated by host or viral enzymes. The steps in viral j replication and the main sites of action of such antiviral drugs are illustrated in Figure V-3-1. j... 

An alternative mechanism of SAB action could involve its known effects on de novo purine biosynthesis (1, S) and/or nucleoside transport (5). The combined inhibitory effects of SAB and purine analogues on purine biosynthesis could result in sufficient depletion of intracellular nucleotide pools to result in enhanced cellular cytotoxicity. In addition, these effects would lead to an increased bioavailability of 5-phosphoribosyl-l-pyrophosphate (PRPP), the first enzymic product in the de novo pathway. Increased PRPP levels would enhance the activity of hypoxanthine phosphoribosyl transferase, leading to increased salvage of purine analogues. 

The specificity of ribonuclease has been studied with small synthetic substrates. When diesters of various nucleotides are subjected to RNAase, only those compounds that are derivatives of pyrimidine nucleoside 3 -phosphate are hydrolyzed purine nucleotides and pyrimidine 2 - or 5 -phosphates are resistant to this enzyme. With both model substrates and ribonucleic acid, the action of RNAase has been shown to include the intermediate formation of cyclic nucleotides (III). ... 

There are several pathways available for the degradation of the mononucleotides. For example, adenosine 5 -phosphatc (AMP) is either deaminated hydrolytically to inosinic acid (IMP) by 6 -adenylic acid deaminase (217, iS7) or split directly to the corresponding nucleoside, adenosine, by 5 -nucleotidase 238). The nucleoside inosine resulted from either the hydrolysis of inosinic acid by 5 -nucleotidase or by the action of adenosine deaminase on adenosine 238, 239). The above pathways, as well as other likely conversions of purine compounds to hypoxanthine and xanthine 2JiO) are shown in Fig. 13. Finally, the enzyme xanthine oxidase acted on both the free bases, hypoxanthine and xanthine, to produce uric acid which was the final product of purine metabolism in some animals. 

---