Membrane-bound enzymes proton gradient

The outcome of these coupled reactions, both reversible under cellular conditions, is that the energy released on oxidation of an aldehyde to a carboxylate group is conserved by the coupled formation of ATP from ADP and Pj. The formation of ATP by phosphoryl group transfer from a substrate such as 1,3-bisphosphoglycerate is referred to as a substrate-level phosphorylation, to distinguish this mechanism from respiration-linked phosphorylation. Substrate-level phosphorylations involve soluble enzymes and chemical intermediates (1,3-bisphosphoglycerate in this case). Respiration-linked phosphorylations, on the other hand, involve membrane -bound enzymes and transmembrane gradients of protons (Chapter 19). 

As the NADH is oxidized, the electrons released are removed by specific carriers, and the protons are transported from cytoplasm to outside the cell. Removal of H+ causes an increase in the nmnber of OH ions inside the membrane. These conditions result in a proton gradient (pH gradient) across the membrane. This gradient of potential energy, termed as proton motive force, can be used to do useful work. This potential energy is captured by the cell by a series of complex membrane-bound enzymes, known as the ATPase in the process called oxidative phosphorylation. In 1961, the concept of proton gradient was first proposed as chemiosmotic theory by Peter Mitchell of England, who won the Nobel Prize for this scientific contribution. 

Membrane-bound ATP synthetase is an enzyme present in all oxidative and/or photosynthetic organisms. It catalyses the formation of ATP from ADP and P using the electrochemical potential (proton gradient) across the membrane by a mechanism which is still not well understood. 

Thylakoid vesicles as normally isolated possess little or no ATPase activity, despite their ability to catalyse vigorous photophosphorylation. Several treatments elicit an ATPase activity which is catalysed by the membrane-bound ATP synthase. Such treatments involve both the imposition of a transmembrane proton electrochemical gradient (A/ah+) and the reduction of a disulfide group on the y subunit of the enzyme [29,30]. 

The blue oxidases are soluble extracellular enzymes (see, e.g., Messerschmidt ) whereas the terminal respiratory oxidases are membrane bound and use the free energy available from this reaction to pump protons across the membrane. The transmembrane proton and voltage gradient generated by the oxidase and other components of the aerobic respiratory chain is converted directly to more useful forms by a number of membrane-bound energy-conserving systems, such as the ATP synthase and secondary active transport systems (see, e.g., Calhoun et... 

The gastric mucosa is able to generate a proton gradient of 10 1. The energy required for this transport can in principle be delivered by an ATPase and a search for such an enzyme was indicated. The earlier suggestion that a membrane-bound anion-sensitive ATPase in cooperation with carbonic anhydrase would be responsible, has been considered in Section 2 and found to be unlikely. 

The membrane-bound ATP(synth)ase affinity (Michaelis constant K ) for its substrates was found quite variable for ADP, from less than T yM (1) to almost 200 yM (2). In fact, this was predictable (3), inasmuch as the chemiosmotic mechanism of phosphorylation makes that, as soon as ADP is added, the proton channels open, the proton gradient A]5f + lowers, and consequently the catalytic constant and/or the enzyme number, because of their AvL+-dependent activation (4), decrease. That is, is not constant in the kinetic determination of K. ... 

Figure 6.7 The part of ATP synthase that is out of the membrane and rotates to form ATP from ADP and free phosphate. Rotation is driven by a proton gradient in the membrane-bound part of the enzyme [adapted from Boyer (1999)].

Matsushita K, Shinagawa E, Ameyama M (1982) o-Gluconate dehydrogenase from bacteria, 2-keto-D-gluconate-yielding, membrane-bound. Methods Enzymol 89 187-193 Matsushita K, Patel L, Kaback HR (1984) Cytochrome o type oxidase from Escherichia coli. Characterization of the enzyme and mechanism of electrochemical proton gradient generation. Biochemistry 23 4703-4714... 

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