Phosphorylation of ADP From

Gramicidin was found to uncouple the phosphorylation of ADP from the enzymatic reduction of ferricytochrome C167,168,169,170,171,172,173,174,175 ... 

Uncoupler. A substance that uncouples phosphorylation of ADP from electron transfer for example, 2,4-dinitrophenol. 

Calculate the number of molecules of ATP that can be produced by substrate-level phosphorylation of ADP from two molecules of acetyl-CoA in the (a) citric acid cycle and (b) glyoxylate cycle. 

Figure 16-2. The citric acid cycle the major catabolic pathway for acetyl-CoA in aerobic organisms. Acetyl-CoA, the product of carbohydrate, protein, and lipid catabolism, is taken into the cycle, together with HjO, and oxidized to CO2 with the release of reducing equivalents (2H). Subsequent oxidation of 2H in the respiratory chain leads to coupled phosphorylation of ADP to ATP. For one turn of the cycle, 11 are generated via oxidative phosphorylation and one arises at substrate level from the conversion of succinyl-CoA to succinate.

The individual steps of the multistep chemical reduction of COj with the aid of NADPHj require an energy supply. This supply is secured by participation of ATP molecules in these steps. The chloroplasts of plants contain few mitochondria. Hence, the ATP molecules are formed in plants not by oxidative phosphorylation of ADP but by a phosphorylation reaction coupled with the individual steps of the photosynthesis reaction, particularly with the steps in the transition from PSII to PSI. The mechanism of ATP synthesis evidently is similar to the electrochemical mechanism involved in their formation by oxidative phosphorylation owing to concentration gradients of the hydrogen ions between the two sides of internal chloroplast membranes, a certain membrane potential develops on account of which the ATP can be synthesized from ADP. Three molecules of ATP are involved in the reaction per molecule of COj. 

It was also observed in earlier studies that mitochondria not only accumulate Ca2+ as an alternative to phosphorylation of ADP (Ca2+ uptake uncouples phosphorylation from electron transport), but could also accumulate much larger amounts of Ca2+ if phosphate was also taken up, resulting in precipitation of Ca2+ within the matrix as insoluble hydroxyapatite, visible as electron-dense granules by EM. An unusual feature of these hydroxyapatite deposits is that they fail to become crystalline and remain amorphous even over protracted periods of time. Their presence in mitochondria in a number of disease conditions underlines the role for mitochondria as a sort of safety device, which can enable the cell to survive, if only for a limited period of time, situations of cytoplasmic Ca2+ overload. 

Pyruvate kinase the last enzyme in aerobic glycolysis, it catalyzes a substrate-level phosphorylation of ADP using the high-energy substrate phosphoenolpyruvate (PEP). Pyruvate kinase is activated by fructose 1,6-bisphosphate from the PFK-1 reaction (feedforward activation). 

This phosphotransferase [EC 2.7.2.1] catalyzes the thermodynamically favored phosphorylation of ADP to form ATP Aeq = [ATP][acetate]/ [acetyl phosphate] [ADP] = 3000). GDP is also an effective phosphoryl group acceptor. This enzyme is easily cold-denatured, and one must use glycerol to maintain full catalytic activity. Initial kinetic evidence, as well as borohydride reduction experiments, suggested the formation of an enzyme-bound acyl-phosphate intermediate, but later kinetic and stereochemicaT data indicate that the kinetic mechanism is sequential and that there is direct in-line phosphoryl transfer. Incidental generation of a metaphosphate anion during catalysis may explain the formation of an enzyme-bound acyl-phosphate. Acetate kinase is ideally suited for the regeneration of ATP or GTP from ADP or GDP, respectively. 

Proton pump Electron transport is coupled to the phosphorylation of ADP by the transport of protons (H+) across the inner mitochon drial membrane from the matrix to the intermembrane space. This process creates across the inner mitochondrial membrane an electrical gradient (with more positive charges on the outside of the membrane than on the inside) and a pH gradient (the outside of the... 

All can yield four molecules of ATP. Sucrose is converted into one molecule each of glucose and fructose each of these sugars requires two molecules of ATP to reach the stage of two molecules of glyceraldehyde 3-phosphate. From here to pyruvate, each glyceraldehyde 3-phosphate molecule yields two molecules of ATP by substrate-level phosphorylation of ADP. Thus, one molecule of glucose or of fructose generates two molecules of ATP. 

The study of bioenergetics involves the study of (1) the processes by which reduced nicotinamide and flavin nucleotides, generated primarily from the oxidation of carbohydrates (Chap. 11) and lipids (Chap. 13), are oxidized ultimately by molecular oxygen via the mitochondrial electron-transport chain, and (2) the mechanism by which this oxidation is coupled to ATP synthesis. The synthesis of ATP in this way is referred to as oxidative phosphorylation, in contrast to phosphorylation of ADP via soluble enzymes. The latter involves intermediate phosphate derivatives of the substrate and is known as substrate-level phosphorylation (Chap. 11). 

Phosphorylation of ADP to ATP by mitochondria is driven by an electrochemical proton gradient established across the inner mitochondrial membrane as a consequence of vectoral transport of protons from NADH and succinate during oxidation by the respiratory chain (see Chapter 17). Hence, lipophilic weak acids or bases (such as 2,4-dinitrophenol) that can shuttle protons across membranes will dissipate the proton gradient and uncouple oxidation from ADP phosphorylation. Intrami-tochondrial ADP can be rate-limiting as demonstrated by inhibition of the mitochondrial adenosine nucleotide carrier by atractyloside. Inhibition of ATP synthesis... 

With these electron transport components, path can be created for the transfer of electrons from substrate to oxygen. Complexes I and IV obviously have enough energy change to support the phosphorylation of ADP to ATP, while complex III is marginal and complex II obviously does not have sufficient energy change. 

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