ATP Regeneration Systems

Murata, K. Uchida, T. Kato, J. Chibata, L Polyphosphate kinase distribution, some properties and its applicatin as an ATP regeneration system. Agric. Biol. Chem., 52, 1471-1477 (1988)... 

Kameda, A. Shiba, T. Kawazoe, Y. Satoh, Y. Ihara, Y. Munekata, M. Ishige, K. Noguchi, T. A novel ATP regeneration system using polyphos-phate-AMP phosphotransferase and polyphosphate kinase. J. Biosci. Bioeng., 91, 557-563 (2001)... 

Moreover, it was recently shown that the deliberate removal of phosphatases from the in vitro translation mixture by immunoprecipitation results in increased protein yields (Shen et al., 1998). As proposed earlier (Jermutus et al., 1998), the elimination of one of the many causes of fast ATP and GTP depletion extends the time of synthesis, and, as a consequence, the total amount of produced protein. It should be possible to similarly remove proteases and nucleases (see above). Together with new ATP regeneration systems to keep the biochemical energy level at a high steady state (Kim and Swartz, 1999), the optimization measures mentioned should increase the level of synthesis of most proteins, and this should directly improve in vitro selection. 

This pathway retains the phosphoanhydride-bound energy of PolyP. The enzymes from A. johnsonii were used to create an ATP-regenerating system (Resnick and Zehnder, 2000). 

Kim, D. M. and J. R. Swartz. Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol Bioeng (1999) 66(3) 180-8. 

CoA and an ATP-regenerating system were added to the mitochondrial incubation (----------------), the... 

The high ATPase activity of broken cell preparations from many tissues makes the use of an ATP-regenerating system mandatory. This may consist of 20 mM phosphoenol pyruvate and 130/ig/0.15 ml of pyruvate kinase [20], which serves to recycle the ADP formed by ATPase back to ATP. 

The ATP-regenerating system may complicate the analysis because of its possible influence on the adenylate cyclase activity. For example, 5 -adenylyl imidodiphosphate (AMP-PNP) labelled with a- P, an analogue of ATP containing Nitrogen substituted for Oxygen between the terminal phosphates, has been used to circumvent the problems encountered in maintaining the substrate concentration during kinetic studies of adenylate cyclase activity [109]. AMP-PNP is a substrate for adenylate cyclase and is only slowly hydrolysed by ATPase. Unlabelled AMP-PNP... 

The most widely used and best developed enzyme-catalyzed phosphorylations are the ones that are coupled to ATP regeneration systems. Sugar phosphates, nucleoside phosphates and glycerides are the major classes of compounds prepared with these methods. 

Adenosine triphosphate (ATP) is the primary energy source for ceU-free synthesis reactions. Rapid depletion of ATP leads to the cessation of translation in under an hour in batch mode. ATP regeneration systems are used to extend the life of translation reactions by maintaining a stable ATP concentration. ATP is regenerated by the enzyme-catalyzed transfer of high-energy phosphate bonds from a secondary energy... 

Figure 5. Hexokinase-catalyzed phosphorylation of fluorinated hexoses coupled with an ATP regeneration system using phosphoenol pyruvate as phosphorylating agent and pyruvate kinase as catalyst.

In this report, we describe the general properties of thermostable enzymes, an ATP regeneration system using thermostable enzymes, and four applications of our thermostable ATP regeneration system. 

Our ATP regeneration system (4,5) is composed of the following five units (Figure 3) 1) a substrate unit (containing AMP, ATP and AcOP), 2) an... 

Synthesis of Fructose 1,6-Diphosphate. Production of FDP is another example of the use of our ATP regeneration system. FDP is used as an intravenous therapeutic agent. It is used in resuscitation, total parenteral nutrition, and cardiology. An Italian company produces more than 20 tons of FDP per year by fermentation. The fermentation process... 

Figure 3. Schematic representation of the ATP regeneration system. PI, P2, P3 and VI, V2, V3 represent peristaltic pumps and electric valves, respectively.

Figure 4. Synthesis of G-6-P using a bioreactor coupled with ATP regeneration system. GIcK = glucokinase AD adenylate kinase.

We can produce F-6-P and FDP from fructose in kilogram-quantities. The catalytic action of hexokinase (E.C. 2.7.1.1), coupled to an ATP regeneration system, converts fructose to fructose 6-phosphate. The action of phosphofructokinase (E.C. 2.7.1.11), again coupled to ATP regeneration, converts fructose 6-phosphate to FDP. 

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