Kinase adenylate

Adenylate kinase (myokinase) catalyzes the following reversible reaction. 

In one assay developed to measure this activity, ion-paired reversed-phase HPLC (Cis) was used to separate the reactants from the products. The mobile phase was 0.065 M potassium phosphate with 1 mM tetrabutylammonium phosphate adjusted to pH 3.6 with phosphoric acid and 3% acetonitrile. The column was eluted isocratically and monitored at 254 nm and with a radio- 

The reaction mixture contained the substrates unlabeled ATP and radioactive [3H]AMP, buffered with Tris-HCl (pH 7.4). The reaction was started by addition of enzyme and terminated by injection of samples directly onto the HPLC column. 

Amyloplast inner membrane 98 Aminoacyl tRNA-synthetase 16 

Adenylate kinase was inactivated by incubation with AP2-PL in a time-dependent 

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The first technique is very intuitive. Out of the few proteins that could be crystallized in a number of different conformations, adenylate kinase is probably the best-studied example. By combining nine observed crystal structures and interpolating between them, a movie was constructed that visualized a hypothetical path of its hinge-bending transition (jVonrhein et al. 1995]). 

To facilitate conformational transitions in the before-mentioned adenylate kinase, Elamrani and co-workers scaled all atomic masses by a large factor thus allowing the use of a high effective simulation temperature of 2000K ([Elamrani et al. 1996]). To prevent protein unfolding, elements of secondary structure had to be constrained. 

Elamrani et al. 1996] Elamrani, S., Berry, M.B., Phillips Jr., G.N., McCammon, J.A. Study of Global Motions in Proteins by Weighted Masses Molecular Dynamics Adenylate Kinase as a Test Case. Proteins 25 (1996) 79-88 [Elcock et al. 1997] Elcock, A.H., Potter, M.J., McCammon, J.A. Application of Poisson-Boltzmann Solvation Forces to Macromolecular Simulations. In Computer Simulation of Biomoleeular Systems, Vol. 3, A.J. Wilkinson et al. eds., ESCOM Science Publishers B.V., Leiden... 

Figure 4.14 Examples of different types of open twisted a/p structures. Both schematic and topological diagrams are given. In the topological diagrams, arrows denote strands of p sheet and rectangles denote a helices, (a) The FMN-binding redox protein flavodoxln. (b) The enzyme adenylate kinase, which catalyzes the reaction AMP +...

The second structure, adenylate kinase (Figure 4.14b), has two such posi-I tions, one on each side of p strand 1. The connection from strand 1 to strand 12 goes to the right, whereas the connection from the flanking strands 3 and 4 both go to the left. Crevices are formed between p strands 1 and 3 and [between strands 1 and 4. One of these crevices forms part of an AMP-binding [site, and the other crevice forms part of an ATP-binding site that catalyzes the Iformation of ADP from AMP and ATP. 

AMP reverses the inhibition due to ATP, and AMP levels in cells can rise dramatically when ATP levels decrease, due to the action of the enzyme adenylate kinase, which catalyzes the reaction... 

Adenylate kinase rapidly interconverts ADP, ATP, and AMP to maintain this equilibrium. ADP levels in cells are typically 10% of ATP levels, and AMP levels are often less than 1% of the ATP concentration. Under such conditions, a small net change in ATP concentration due to ATP hydrolysis results in a much larger relative increase in the AMP levels because of adenylate kinase activity. 

The problem can be solved using the equilibrium expression for the adenylate kinase reaction ... 

Mitochondria are surrounded by a simple outer membrane and a more complex inner membrane (Figure 21.1). The space between the inner and outer membranes is referred to as the intermembrane space. Several enzymes that utilize ATP (such as creatine kinase and adenylate kinase) are found in the intermembrane space. The smooth outer membrane is about 30 to 40% lipid and 60 to 70% protein, and has a relatively high concentration of phos-phatidylinositol. The outer membrane contains significant amounts of porin —a transmembrane protein, rich in /3-sheets, that forms large channels across the membrane, permitting free diffusion of molecules with molecular weights of about 10,000 or less. Apparently, the outer membrane functions mainly to... 

The lower panel shows the decreasing concentration of ATP, to about 60% of resting levels, and the simultaneous equimolar increase in IMP. The fall in ATP started when most of the PCr store was utilized, resulting in a decreased rate of ADP phosphorylation via the creatine kinase reaction. The resultant accumulation of ADP stimulates adenylate kinase activity and subsequently IMP is formed via the AMP deaminase reaction ... 

Adenosine 5 -hypophosphate (23), an analogue of ADP, can undergo phosphorylation by PEP and pyruvate kinase to yield (24). Adenylate kinase which catalyses the scission of the bond between the a and j8 phosphorus atoms in ADP is, not surprisingly, inhibited competitively by (23). 

Comparison with the nucleotide binding sites in adenylate kinase... 

Maker, H.S. Weiss, C. and Brannan. Amine-mediated toxicity The effects of dopamine, norepinephrine, 5-hydroxytryptamine, 6-hydroxy-dopamine, ascorbate, glutathione and peroxide on the in vitro activities of creatine and adenylate kinases in the brain of the rat. 

Adenylate kinase (AK) is a ubiquitous monomeric enzyme that catalyzes the interconversion of AMP, ADP, and ATP. This interconversion of the adenine nucleotides seems to be of particular importance in regulating the equilibrium of adenine nucleotides in tissues, especially in red blood cells. AK has three isozymes (AK 1,2, and 3). AK 1 is present in the cytosol of skeletal muscle, brain, and red blood cells, and AK 2 is found in the intermembrane space of mitochondria of liver, kidney, spleen, and heart. AK 3, also called GTP AMP phosphotransferase, exists in the mitochondrial matrix of liver and heart. 

B15. Beutler, E., Carson, D., Dannawi, H., Forman, L., Kuhl, W., West, C., and Westwood, B., Metabolic compensation for profound erythrocyte adenylate kinase deficiency A hereditary enzyme defect without hemolytic anemia. J. Clin. Invest. 72,648-655 (1983). 

B33. Boivin, P, Galand, C Hakim, J., Simony, J., and Seligman, M., Une nouvelle erythroenzy-mopathies Anemie hemolytique congenitale non spherocytaire et deficit hereditaire en adenylate-kinase erythrocytaire. Presse Med. 79,215-218 (1971). 

M14. Matsuura, S., Igarashi, M Tanizawa, Y., Yamada, M., Kishi, F Kajii, T Fujii, H., Miwa, S Sakurai, M., and Nakazawa, A., Human adenylate kinase deficiency associated with hemolytic anemia. A single base substitution affecting solubility and catalytic activity of cytosolic adenylate kinase. J. Biol. Chem. 264, 10148-10155 (1989). 

M25. Miwa, S., Fujii, H., Tani, K., Takahashi, K Takizawa, T., and Igarashi, T Red cell adenylate kinase deficiency associated with hereditary nonspherocytic hemolytic anemia Clinical and biochemical studies. Am. J. Hematol. 14,325-333 (1983). 

Szeinbeig, A., Kahana, D., Slava, G., Zaidman, J., and Ben-Ezzer, B Hereditary deficiency of adenylate kinase in red blood cells. Acta Haematol. 42,111-126 (1969). 

T18. Toren, A., Brok-Simoni, F Ben-Bassat, I., Holtman, F., Mandel, M., Neumann, Y., Ramot, B., Rechavi, G and Kende, G., Congenital haemolytic anaemia associated with adenylate kinase deficiency. Br. J. Haematol. 87,376-380 (1994). 

For luciferin, a firefly luciferase cosubstrate, another method of retention has been evaluated which consisted of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a polymeric matrix31. Using the suitable co-immobilized enzymes (adenylate kinase and creatine kinase), the three adenylic nucleotides (ATP, ADP and AMP) could be assayed continuously and reproducibly with a selfcontainment working time of 3 h. 

The G-protein serves as a timekeeper and binds GTP, and with GTP bound, it can couple the receptor and activate the adenylate cyclase. However, the G-protein slowly hydrolyzes the bound GTP to GDP and Pj. When this happens, the whole complex falls apart and the adenylate kinase is inactivated. 

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