Electron phosphoryl group transfer

Stabilization of enolate anions generated from abstraction of a proton a to a carboxylate Hydrolysis, phosphoryl group transfer via hydrolytic nucleophilic substitution Stabilization of diverse oxyanion intermediates via metal-assisted catalysis Schiff base dependent formation of an electron sink ... 

Inhibition of ATP synthase (energy transfer) reduces proton flow from the inter-membrane space to the matrix, which inhibits electron flow in the respiratory chain. Oligomycin, a macrolide antibiotic, prevents phosphoryl group transfer of ATP synthase. Dicyclohexylcarbodimide (DCCD) binds to and inhibits ATP synthase. Similar to the inhibitors of Complexes I, III, and IV, energy transfer inhibitors cause accumulation of reactive electrons and generate ROS. 

Describe the structural and electronic bases for the high phosphoryl group-transfer potential of ATP, and give the free energy liberated by the hydrolysis of ATP under standard and cellular conditions. 

The transfer of phosphoryl groups is a central feature of metabolism. Equally important is another kind of transfer, electron transfer in oxidation-reduction reactions. These reactions involve the loss of electrons by one chemical species, which is thereby oxidized, and the gain of electrons by another, which is reduced. The flow of electrons in oxidation-reduction reactions is responsible, directly or indirectly, for all work done by living organisms. In nonphotosynthetic organisms, the sources of electrons are reduced compounds (foods) in photosynthetic organisms, the initial electron donor is a chemical species excited by the absorption of light. The path of electron flow in metabolism is complex. Electrons move from various metabolic intermediates to specialized electron carriers in enzyme-catalyzed reactions. 

In the overall glycolytic process, one molecule of glucose is converted to two molecules of pyruvate (the pathway of carbon). Two molecules of ADP and two of Pi are converted to two molecules of ATP (the pathway of phosphoryl groups). Four electrons, as two hydride ions, are transferred from two molecules of glyceralde-hyde 3-phosphate to two of NAD+ (the pathway of electrons). 

An interesting experiment is to allow oxidative phosphorylation to proceed until the mitochondria reach state 4 and to measure the phosphorylation state ratio Rp, which equals the value of [ATP] / [ADP][PJ that is attained. This mass action ratio, which has also been called the "phosphorylation ratio" or "phosphorylation potential" (see Chapter 6 and Eq. 6-29), often reaches values greater than 104-105 M 1 in the cytosol.164 An extrapolated value for a zero rate of ATP hydrolysis of log Rf) = 6.9 was estimated. This corresponds (Eq. 6-29) to an increase in group transfer potential (AG of hydrolysis of ATP) of 39 kj/mol. It follows that the overall value of AG for oxidation of NADH in the coupled electron transport chain is less negative than is AG. If synthesis of three molecules of ATP is coupled to electron transport, the system should reach an equilibrium when Rp = 106 4 at 25°C, the difference in AG and AG being 3RT In Rp = 3 x 5.708 x 6.4 = 110 kj mol-1. This value of Rp is, within experimental error, the same as the maximum value observed.165 There apparently is an almost true equilibrium among NADH, 02 and the adenylate system if the P/O ratio is 3. 

However, if the electron transport between 3-hydroxybutyrate and cytochrome b562 is tightly coupled to the synthesis of one molecule of ATP, the observed potential of the carrier will be determined not only by the imposed potential E of the equilibrating system but also by the phosphorylation state ratio of the adenylate system (Eq. 18-7). Here AG atp is the group transfer potential (-AG of hydrolysis) of ATP at pH 7 (Table 6-6), and n is the number of electrons passing through the chain required to synthesize one ATP. In the upper part of the equation n is the number of electrons required to reduce the carrier, namely one in the case of cytochrome b562. 

The activating effect of Mg2+ upon the cleavage of the phosphoryl group from the ATP could reflect the enhancement of an SN2 reaction at phosphorus by electron withdrawal and charge neutralization via coordination to the metal (equation 1). Support for an SN2 mechanism comes from a consideration57 of the inhibition by vanadate. Coordination of the transferable phosphoryl group would inhibit the SN1 mechanism. 

The side chains of amino acids present a number of nucleophilic groups for catalysis these include RCOO-, R—NH2, aromatic—OH, histidyl, R—OH, and RS. These groups attack electrophilic (electron-deficient) parts of substrates to form a covalent bond between the substrate and the enzyme, thus forming a reaction intermediate. This type of process is particularly evident in the group-transfer enzymes (EC Class 2 see Table 8.1). In the formation of a covalently bonded intermediate, attack by the enzyme nudeophile (Enz-X in Example 8.10) on the substrate can result in acylation, phosphorylation, or glycosylation of the nucleophile. 

The hydrolysis of a thioester is thermodynamically more favorable than thai of an oxygen ester because the electrons of the C=0 bond cannot form resonance structures with the C—S bond that are as stable as those that they can form with the C—O bond. Consequently, acetyl CoA has a high acetyl group-transfer potential because transfer of the acetyl group is exergonic. Acetyl CoA carries an activated acetyl group, just as ATP carries an activated phosphoryl group. 

Protonation, metal coordination, or further esterification of the transferable phosphoryl group itself, profoundly retards the Sul mechanisms (3), presumably because of the decreased availability of lone pair electrons of oxygen for n bonding to phosphorus as metaphosphate is expelled, but accelerates the Sj 2 mechanism by charge neutralization (6), and possibly by electron withdrawal from phosphorus. In addition, metal coordination could accelerate Sn2 displacements by five additional mechanisms (5). Chelation of the phosphoryl group could... 

A very clever three-phase test for the detection of metaphosphate intermediates in phosphoryl transfer reactions has been described by Rebek and coworkers (44). The basis of this test is the use of two polymers suspended in solution. The donor polymer contains a potential precursor to metaphosphate anion, e.g., an acyl phosphate or a phosphoramidate, and the recipient polymer contains an acceptor nucleophile, e.g., an amine. After reaction and physical separation of the polymers, the recipient polymer is analyzed for covalently bound phosphate. Since very few of the phosphoryl groups to be transferred will be on the surface of the donor polymer, detection of significant transfer to the recipient polymer provides evidence for a diffusible intermediate, i.e., free metaphosphate anion. Significant transfer did occur in dioxane or acetonitrile suspensions of the polymers, thereby providing evidence for an intermediate. However, this test for diffusible and, therefore, relatively stable metaphosphate anion is compromised by the choice of solvent. Both dioxane and acetonitrile can provide unshared electron pairs for the highly electrophilic metaphosphate anion such that the actual species that migrates from the donor polymer to the recipient polymer may be a complex between metaphosphate anion and the solvent. Such a role for solvent has been investigated stereochemically, the results of which will be described later in this section. 

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