High-energy intermediate activators

The mechanism of succinyl-CoA synthetase is postulated to involve displacement of CoA by phosphate, forming succinyl phosphate at the active site, followed by transfer of the phosphoryl group to an active-site histidine (making a phosphohistidine intermediate) and release of succinate. The phosphoryl moiety is then transferred to GDP to form GTP (Figure 20.13). This sequence of steps preserves the energy of the thioester bond of succinyl-CoA in a series of high-energy intermediates that lead to a molecule of ATP ... 

Many models were proposed to account for the coupling of electron transport and ATP synthesis. A persuasive model, advanced by E. C. Slater in 1953, proposed that energy derived from electron transport was stored in a high-energy intermediate (symbolized as X P). This chemical species—in essence an activated form of phosphate—functioned according to certain relations according to Equations (21.22)-(21.25) (see below) to drive ATP synthesis. 

The plasma membrane Ca2+-ATPase pump effects outward transport of Ca2+ against a large electrochemical gradient for Ca2+. The mechanism of the pump involves its phosphorylation by ATP and the formation of a high-energy intermediate. This basic mechanism is similar for both the plasma membrane and ER pumps however, the structures of these distinct gene products are substantially different. As discussed below, the ER pump, sometimes called a sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, is inhibited potently by certain natural and synthetic toxins that do not affect the plasma membrane pump. The plasma membrane pump, but not the SERCA pump, is controlled in part by Ca2+ calmodulin, allowing for rapid activation when cytoplasmic Ca2+ rises. 

Recentiy published crystal structures of antibody 4C6, an antibody that catalyzes another cationic cyclization reaction (Figure 6), revealed that this antibody has exquisite shape complementarity to its eliciting hapten 5. The active site contains multiple aromatic residues which shield the high-energy intermediate from solvent and stabilize the carbocation intermediates through cation-7r interactions. 

Sucdnyl CoA is a high-energy intermediate that can be used for heme synthesis and to activate ketone bodies in extrahepatic tissues. 

Therefore, this method allows for the determination of relative rate constants for the excitation step in a complex reaction system, where this step cannot be observed directly by kinetic measurements. The singlet quantum yield at infinite activator concentrations (high-energy intermediates formed interact with the activator, is also obtained from this relationship (equation 5). 

In this part of the chapter, we will focus essentially on mechanistic aspects of the peroxyoxalate reaction. For the discussion of the most important advances in mechanistic aspects of this chemiluminescent system, covering mainly literature reports published in the last two decades, we will divide the sequence operationally into three main parts (i) the kinetics of chemical reactions that take place before chemiexcitation, which ultimately produce the high-energy intermediate (HEI) (ii) the efforts to elucidate the structure of the proposed HEIs, either attempting to trap and synthesize them, or by indirect spectroscopic studies and lastly, (iii) the mechanism involved in chemiexcitation, whereby the interaction of the HEI with the activator leads to the formation of the electronically excited state of the latter, followed by fluorescence emission and decay to the ground state. 

Hexanal, hpid oxidation assessment, 669 1-Hexene, primary ozonide, 720 High-density lipoprotein (HDL) oxidation, 612 TEARS assay, 667 High-energy intermediate (HEI) chemiluminescence, 1215 activators, 1220, 1222 peroxyoxalates, 1188-9, 1257, 1261-6, 1267, 1269 stmcture, 1262, 1263... 

An indirect method has been used to determine relative rate constants for the excitation step in peroxyoxalate CL from the imidazole (IM-H)-catalyzed reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) with hydrogen peroxide in the presence of various ACTs18. In this case, the HEI is formed in slow reaction steps and its interaction with the ACT is not observed kinetically. However, application of the steady-state approximation to the reduced kinetic scheme for this transformation (Scheme 6) leads to a linear relationship of 1/S vs. 1/[ACT] (equation 5) and to the ratio of the chemiluminescence parameters /ic vrAi), which is a direct measure of the rate constant of the excitation step. Therefore, this method allows for the determination of relative rate constants for the excitation step in a complex reaction system, where this step cannot be observed directly by kinetic measurements18. The singlet quantum yield at infinite activator concentrations ( °), where all high-energy intermediates formed interact with the activator, is also obtained from this relationship (equation 5). 

The chemical mechanism of activation (scheme 1) involves the nucleophilic attack of the carboxylate of Tyr on the a-phosphate of ATP to generate either a 5-coordinate transition state or a high energy intermediate. This then expels PPj... 

Conjugation reactions usually involve metabolite activation by some high-energy intermediate and have been classified into two general types type I, in which an activated conjugating agent combines with the substrate to yield the conjugated product, and type II, in which the substrate is activated and then combines with an amino acid... 

The intermediates and their relative energies for the uncatalyzed hydrolysis of 4.1 are shown in Figure 4.1. The first step, a proton transfer, is unfavorable because both the acid (4.2) and base (4.1) are weak. The first step, therefore, is endothermic, has a high energy of activation, and is very slow. Other uncatalyzed mechanisms are possible, but all involve an energetically unfavorable step at some point along the pathway. 

The substrate water molecules are prepared in a stepwise fashion for 0-0 bond formation by binding to the M OjCa cluster and by (partial) deprotonation. The concerted oxidation of the activated substrate occurs then either in two 2e steps or in one concerted 4e reaction step, thus avoiding high-energy intermediates. It is the matrix (protein) and the Ca2+/CF ions that allow for the coupling of proton- and electron-transfer reactions to occur. These features are... 

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