Substrate-linked phosphorylation

A model reaction that supports the above mechanism is the glycolytic substrate-linked phosphorylation, which proceeds via a thiol ester prior to the formation of the phosphorylated intermediate (Chapter 13). Although the chemical hypothesis is consistent with the substrate-linked phosphorylation mechanism, it is deficient in explaining the oxidative phosphorylation in mitochondria for two reasons ... 

Severo s laboratory was also possibly for some of us unfortunate in that it focused thinking on the mechanism of substrate-linked phosphorylation . 

Another important function of the kinases is the synthesis of ATP from ADP and energy-rich bound phosphate in substrate-linked phosphorylation (cf. Chapt. XV-7) and in respiratory-chain phosphorylation (Chapt. X-6). ATP hence represents a pool for energy-rich phosphates—and, in a sense, for chemical energy in general. 

The preceding paragraphs undoubtedly have revealed the complicated and diverse nature of carbohydrate metabolism, both on the level of interconversions among the carbohydrates and on that of degradative reactions for the production of energy. Part of the energy is derived anaerobically by substrate-linked phosphorylation the major part, however, is liberated in the respiratory chain. The situation is further complicated by the obvious fact that carbohydrate metabolism is not an isolated system of reactions, but is closely tied to other pathways and reaction cycles through common intermediates. A separate chapter (Chapt. XVIII) is devoted to such interrelationships. 

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). 

If energy is generated solely by substrate-level phosphorylation, as with anaerobic fermentative metabolism of bacteria and yeast, then the yield is more tightly linked to the amount of energy generated. Generally 10 to 12 kg of cell dry matter can be synthesised per kmol of ATP generated in metabolism. 

Afterwards, the enzyme glyceraldehyde-3-phosphate dehydrogenase transforms glyceraldehyde-3-phosphateinto 1,3-diphosphoglycerate. This reaction involves the oxidation of the molecule that is linked to reducing NAD+ to NADH in order to redress the redox balance. Simultaneously, a substrate level phosphorylation takes... 

The mechanism of phosphorylation of ADP during glycolysis is quite different from that which takes place in the mitochondria. In glycolysis, the phosphate transfer occins by direct interaction between a metabolite and ADP and is called substrate-level phosphorylation, on the other hand, in mitochondria ATP synthase mediates the reaction in the process of oxidative phosphorylation. This reaction is closely linked to the consumption of oxygen in mitochondria (see Sec. 10.8). 

The breakdown of succinyl coenzyme A may also be linked to the phosphorylation of GDP and IDP to yield GTP and ITP in the presence of inorganic phosphate. The enzyme catalyzing that reaction has been named the phosphorylating enzyme, and has been prepared in a crude form from heart muscle. This preparation also contains another enzyme, nucleoside diphosphate kinase, which catalyzes the transfer of phosphorus from GTP or ITP to ADP to yield ATP. The phosphorylation of ADP coupled to the oxidation of a-ketoglutarate is the only substrate level phosphorylation in the Krebs cycle, and, as can be expected, it is not inhibited by dinitrophenol. When O-labeled phosphate is used in the reaction, the label appears... 

Although the cell contains several enzymic systems generating energy, most cellular energy is provided by the intramitochondrial oxidation of the Krebs cycle substrates linked to the phosphorylation of ATP in the electron transport chain. In addition to their role in oxidative phosphorylation, mitochondria are actively involved in ion transport. The ion exchange is discussed in other parts of this book [163-174]. 

As discussed in section 10.2.2, the formation of fructose bisphosphate, catalysed by phosphofructokinase, is an important step for the regulation of glucose metabolism. Once it has been formed, fructose bisphosphate is cleaved into two three-carbon compounds, which are interconvertible. The metabolism of these three-carbon sugars is linked to both the reduction of NAD" to NADH and direct (substrate-level) phosphorylation of ADP to ATP (section 3.3). The result is the formation of 2 mol of pyruvate from each mole of glucose. 

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

After their synthesis (translation), most proteins go through a maturation process, called post-translational modification that affects their activity. One common post-translational modification of proteins is phosphorylation. Two functional classes of enzymes mediate this reversible process protein kinases add phosphate groups to hydroxyl groups of serine, threonine and tyrosine in their substrate, while protein phosphatases remove phosphate groups. The phosphate-linking... 

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