Potato nucleotide pyrophosphatase

Nucleoside Pyrophosphates. - 2.3.1 Nucleoside Diphosphate Analogues. A large variety of esters with different nucleoside and alkyl moieties (llla-j) have been synthesised in small amounts using different combinations of nucleoside triphosphate, alcohols and snake venom pyrophosphatase. Potato tube pyrophosphatase was also reported as being a possible practical biocatalyst to synthesise such nucleotide pyrophosphate-O-alkyl esters, but using more stringent reaction requirements than that of the snake venom enzyme. ... 

Structure of Coemyme A. The elucidation of the structure of CoA depended heavily on d radation by specific enzymes. The phosphate on carbon 3 of the adenosine was shown to be a monoester phosphate by hydrolysis with prostate phosphomonoesterase. The localization of the monoester at the 3 position was established by its sensitivity to a b nucleotidase that attacks only nucleoside 3 -pbosphates, not 2 - or 5 -phosphates. The original CoA molecule or the phosphatase product, depbospho CoA, can be split by nucleotide pyrophosphatases from potato or snake venom. These reactions permitted the identification of the adenosine phosphate portion of the molecule. The position of the phosphate on pantothenic acid cannot be determined enzymatically, but was established by studies on the synthesis of CoA from synthetic phos-phorylated pantetheines. Pantetheine is split to thiolethanolamine and pantothenic acid by an enzyme found in liver and kidney. This enzyme also attacks larger molecules, including CoA. 

Nucleotide Pyrophosphatase. Nucleotide pyrophosphatase was first detected in particles from animal cells, and was subsequently purified from potato tubers. This enzyme, which has been found widely distributed, splits DPN, TPN, and other dinucleotides with a pyrophosphate bridge. As would be expected from the broad specificity, reduction... 

To determine the structure of arylazido- 3-alanine NAD, the analog is treated with nucleotide pyrophosphatase isolated from potatoes as described by Kornberg and Pricer. After incubation of the arylazido-/8-alanine NAD with pyrophosphatase, the products are separated on Whatman No. 1 filter paper with n-butanol-water-acetic acid (5 3 2). A single UV absorbing spot with Rf value of 0.18 and two orange spots with Rf values of 0.60 and 0.91 were evident (Table III). The orange material with the highest Rf value (0.91) was characteristic of arylazido-yS-alanine. 

TPN differs from DPN in having one more phosphate group, which has been assigned to carbon 2 of the adenylic acid moiety. This is based on several lines of evidence. By splitting the pyrophosphate bond of TPN with potato nucleotide pyrophosphatase it was possible to separate a diphosphoadenosine fragment (compound XI) as the lead salt. Phosphate esterified to carbon 5 was hydrolyzed by an adenosine-5-phosphatase from potato, and the product was found to be identical with adenylic acid a, presumably adenosine-2 -phosphate. Confirmatory evidence has been provided by finding a deaminase active with adenosine-3 -phosphate, adenosine-5 -phosphate, ATP, and DPN but inert with adenosine-2 -phosphate and TPN. ... 

However both can be cleaved by alkali to form UMP and the phosphate derivative of the carbohydrate. The pyrophosphate bond may also be split by a nucleotide pyrophosphatase from potatoes (338) or kidney (339). The enzyme is effective on DPN, DPNH, TPN, ADP, ATP, thiamine pyrophosphate, and FAD. 

It appears that most nucleotide pyrophoqihatases from mammalian tissues cleave the reduced forms of the coenzymes faster than the oxidized nucleotides. The plant nucleotide pyrophosphatases, however, q>lit DPNH, TPNH, and the oxidized coenzyme forms at equal rates. It should be pointed out, however, that an enzyme such as the potato nucleotide pyrophosphatase also attacks ATP and adenosine diphosphate (ADP), whereas the purified pigeon liver enzyme does not. The snake venom enzyme seems to split the oxidized and reduced nucleotide at equal rates. 

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