VOL. 4 (1950) PURINES AND PYRIMIDINES IN RIBOSIDIC LINKAGE 233 



ENZYMATIC SYNTHESIS OF PURINE RIBO-NUCLEOSIDES 



The presence in animal tissues of an enzyme, called nucleosidase which splits of 

 purines from purine nucleosides of the ribose series has been known for many years. 

 Klein^ who made a detailed study of this enzyme found that phosphate and arsenate 

 enhance the enzymatic splitting of purine nucleosides. When I spent some time in 1943- 

 1944 isolating nucleosidases from liver it was done only with the purpose of using these 

 enzymes as analytical tools in an optical micromethod which I was trying to develop 

 at that time. I had no knowledge about Klein's work at the time when I came across 

 the observation that nucleosidase subjected to prolonged dialysis loses its activity. In 

 view of observations by Meyerhof and Cori it was not too far-fetched to try to add 

 inorganic ortho-phosphate to the system and it turned out that this addition completely 

 restored the catalytic activity of the system. Pursuing the analogy to Cori's work on 

 the polysaccharide phosphorylase^ I attempted to demonstrate the formation of ribose- 

 1-phosphate as a suspected intermediate. These attempts failed quite a few times. For- 

 tunately LowRY who was my colleague at that time at The Public Health Research 

 Institute had worked out a new method for phosphate determination which operates 

 at Ph 4- This method, the well-known Lowry-Lopez method^, permits an estimation 

 of highly labile phosphoric esters such as phosphocreatine and acylphosphates in the 

 presence of inorganic phosphate. With the Lowry-Lopez procedure it became possible 

 to show a clearcut proportionality between liberation of purine and uptake of inorganic 

 phosphate*. It was fairly obvious therefore that a new and highly acid-labile phosphoric 

 ester was formed as a product of the enzymatic phosphorolysis of nucleosides. The ester 

 was later obtained as the barium salt. It contained i mole pentose for each mole of 

 labile phosphate and for each equivalent of aldose liberated upon mild acid hydrolysis. 

 Lowry has investigated the lability of ribose-1-phosphate in dilute hydrochloric acid 

 at room temperature and found that 50% of the ester was split after 2.5 minutes incuba- 

 tion in N hydrochloric acid. In view of these properties and the resynthesis experiments 

 described below the new ester was named ribose-1-phosphate. 



The next step was an attempt to resynthesize purine nucleosides with ribose-1- 

 phosphate. This was performed by incubating hypoxanthine, ribose-1-phosphate and 

 a fractionated sample of liver nucleosidase about 20 minutes at 25° and subsequently 

 analysing free and incorporated hypoxanthine^. It was then found that a large propor- 

 tion of the hypoxanthine was incorporated in ribosidic linkage and an equimolar amount 

 of labile phosphate was liberated. This enzymatic synthesis of inosine (ribose-l-hypo- 

 xanthine) proceeded very far; thus, if equimolar amounts of hypoxanthine and ribose- 

 1-phosphate were incubated with the enzyme about 80% of the phosphoriboside was 

 converted into purine-riboside. If the mixture contained twice as much phosphoriboside 

 as hypoxanthine more than 95% of the latter was incorporated in ribosidic hnkage. 

 The equilibrium can be formulated as follows : ribose-1-phosphate ~ h^'poxanthine ^ 

 ribose-1-hypoxanthine -j- phosphate. The enzyme catalysing this equilibrium was named 

 nucleoside phosphorylase. Nucleoside phosphorylase possesses a certain specificity with 

 regard to the nitrogenous bases added as well as to the pentoses present. Inosine and 

 guanine riboside are the only ribosides which undergo phosphorolysis in the presence 

 of the enzyme used. Adenosine and xanthosine are inert in this system as are pyrimidine 

 ribosides. Likewise hypoxanthine and guanine are the only nitrogenous bases which are 

 incorporated, i.e., which in the presence of the enzyme undergo an exchange with the 

 References p. 2J7. 



