Cyclic-2 .3 -ribose phosphate

Top row schematic representation of the hydrolysis of the phospho-ester bond as catalysed by a phosphatase with a histidine in the active site, including the pentacoordinated intermidiate state. Bottom row the two-step mechanism for the cleavage of the phospho-diester bond by ribonucleases, showing the transphosphorylation to cyclic ribose phosphate (step 1) and hydrolysis (step 2). 

Fig. 6.9 Reactions of ADP ribosyl cyclase. Structures of NADP, nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose phosphate (cADPRP). ADP-ribosyl cyclase, in base ex-...

Having defined the oxidative steps for hexosemonophosphate metabolism in yeast, we asked whether these reactions could also be demonstrated in animal tissues. E. J. Seeg-miller, who joined my group in 1950, used our coupled system to study the oxidation of 6-phosphogluconate with rat liver extracts. Not only did he identify ribulose and ribose phosphates as products of the reaction, but he also observed that with continued incubation the pentose phosphates disappeared, and in their place hexosemonophosphate accumu-lated.f > This was the first indication that we were dealing with a cyclic process, in which pentose phosphate formed by the oxidation of hexosemonophosphate could be converted... 

The hnal rehned structure showed a 2, 3 -cyclic phosphate product at the Ci7 ribose position and a Cn nucleotide that had moved dramatically and become almost perpendicular to the Watson-Crick faces of conserved nucleotides Gs and Ae in the catalytic pocket. Interactions of functional groups of G5 and Ae with the product and Cn included (1) a hydrogen bond formed between the exocyclic amine (Ne) of Ag and the cyclic phosphate nonbridging oxygen... 

The nucleotide cyclic AMP (3, 5 -cyclic adenosine monophosphate, cAMP) is a cyclic phosphate ester of particular biochemical significance. It is formed from the triester ATP by the action of the enzyme adenylate cyclase, via nucleophilic attack of the ribose 3 -hydroxyl onto the nearest P=0 group, displacing diphosphate as leaving group. It is subsequently inactivated by hydrolysis to 5 -AMP through the action of a phosphodiesterase enzyme. 

The mechanism involves participation of the free 2 -OH of the ribose groups and formation of cyclic 2, 3 -phosphates and is similar to that of pancreatic ribo-nuclease (Chapter 12). Because deoxyribose lacks the free 2 -OH, the phosphodiester linkages in DNA are quite stable in base. 

AH fungal RNases (T, T , Ni, Ui, and U2) treated in this section catalyze the reaction shown in Fig. 1. The first step (phosphate transfer) is the cleavage of the phosphodiester bond between the 3 and 5 positions of the ribose moities in the RNA chain with the formation of nucleoside 2, 3 -cyclic phosphates and oligonucleotides with 2, 3 -cyclic phosphate at 3 terminal. The nature of the phosphodiester bonds to be cleaved depends on the base specificity of the enzyme. This phosphoryl transfer step is reversible. In the second step (hydrolysis), these terminal cyclic phosphate groups are hydrolyzed with the formation of corresponding 3 -phosphates. Because the first-step is usually faster than the second step, more or less accumulation of the cyclic phosphate may be observed. 

Holy and Sorm (49, 50) observed that RNase T2, like RNase Ti, attacks 9-(/3-D-ribofuranosyl) and 9-(a-L-lyxofuranosyl) derivatives but not 9-(j8-L-ribofuranosyl) and 9-(a-D-lyxofuranosyl) derivatives. Also, like RNase RNase T2 is quite inactive on the phosphodiester bonds of the nucleotide with 2 -0-methyl ribose, such as 2 -0-methyl guanylic acid (96) or 2 -0-methyl cytidylic acid (86). Thus, the action of RNase T2 is in good accord with that of RNase Ti and RNase A on sugar specificity, which may be a common property throughout all RNases, producing 3 -phosphate via 2, 3 -cyclic phosphate. 

The carbohydrate moiety of RNA is D-ribose with the / -D-ribofurano-side ring. The 2 - and 3 -OH groups are cis to each other and easily form the cyclic phosphate intermediate. Although the 2 -deoxynucleotides bind to the enzyme, they only serve as inhibitors. The 2 -OH group is mandatory for the catalytic activity of pancreatic RNase. 

The sugar configuration about the T, 2, 3, and 4 positions can be changed by synthesis. A variety of pyrimidine nucleoside cyclic phosphates have been made. Ukita et al. (425) prepared -D-lyxo-uridine 2 3 -cyclic phosphate (Fig. 21a). The configuration about the 2 and 3 positions is inverted and the two OH groups are now cis to the base rather than trans as in the D-ribose series. No hydrolysis at all of this compound was observed in the presence of RNase. However, both the cyclic phosphate and the free 2 (3 )-nucleotides inhibit the enzyme. The... 

In Fig. 20, B, R, B2, and R2 are the positions of the base and ribose components of the dinucleotide or independent pyrimidine and purine nucleotides, respectively. The phosphate position pi can be occupied by the 3, 5"-diester (5" refers to the 5 position of R2 in a diester) or the 3 - and 5 -nucleotides, respectively. In the protein crystal a sulfate ion occupied this position in variable degree depending on the pH. Histidine 119 can be in any one of four or more positions depending on various factors. The second base might be in position B2 when it is a pyrimidine. The phosphate of a cyclic substrate or pentacovalent intermediate may be at p,. The position labeled H20 is the position of an isolated peak on the electron density map which is interpreted to be a water molecule, Wi, present in the protein and in the complexes. 

Cyclic Pentamers. In uridine-3 -phosphate monohydrate [URIDMP10], the cyclic pentamer configuration involves )NH OH, OH- -OC CH 0(H)P, P-OH- OwH and OwH- -0=C hydrogen bonds, all of which are short (Fig. 17.11 a). It is a homodromic cycle if we include the polarizability of the ribose-0(4 )-CH-component, and probably stabilized by the strong donor/acceptor p-OH of the neutral phosphate group. 

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