Silent substrates

Proteases are enzymes that break peptide bonds in proteins. As such they lend themselves to a variety of homogeneous assay techniques. Most employ labeling both ends of the substrate with a different tag, and looking for the appearance (disappearance) of the signal generated in the intact substrate (product). As an example, for a fluorescence quench assay, the N-terminal of a peptide is labeled with DNP and the C-terminal with MCA. As such, the peptide is fluorescently silent since the fluorescence from DNP is quenched by absorption by the MCA. Another very popular donor/acceptor pair is EDANS 5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and DABCYL 4-(4-dimethylaminophenylazo)benzoic acid) (a sulfonyl derivative (DABSYL) [27], Upon peptide cleavage, the two products diffuse, and due to a lack of proximity, the fluorescence increases. 

Adrenal medulla. On the one hand, release of epinephrine elicits cardiovascular effects, such as increases in heart rate und peripheral vascular resistance. On the other, it evokes metabolic responses, such as glycogenolysis and li-polysis, that generate energy-rich substrates. The sensation of hunger is suppressed. The metabolic state corresponds to that associated with physical exercise - silent stress . 

The sirtuins (silent information regulator 2-related proteins class III HDACs) form a specific class of histone deacetylases. First, they do not share any sequence or structural homology with the other HDACs. Second, they do not require zinc for activity, but rather use the oxidized form of nicotinamide adenine dinucleotide (NAD ) as cofactor. The reaction catalyzed by these enzymes is the conversion of histones acetylated at specific lysine residues into deacetylated histones, the other products of the reaction being nicotinamide and the metabolite 2 -0-acetyl-adenosine diphosphate ribose (OAADPR) [51, 52]. As HATs and other HDACs, sirtuins not only use acetylated histones as substrates but can also deacetylate other proteins. Intriguingly, some sirtuins do not display any deacetylase activity but act as ADP-ribosyl transferases. 

Hemocyanin [30,31], tyrosinase [32] and catechol oxidase (2) [33] comprise this class of proteins. Their active sites are very similar and contain a dicopper core in which both Cu ions are ligated by three N-bound histidine residues. All three proteins are capable of binding dioxygen reversibly at ambient conditions. However, whereas hemocyanin is responsible for O2 transport in certain mollusks and arthropods, catechol oxidase and tyrosinase are enzymes that have vital catalytic functions in a variety of natural systems, namely the oxidation of phenolic substrates to catechols (Scheme 1) (tyrosinase) and the oxidation of catechols to o-quinones (tyrosinase and catechol oxidase). Antiferromagnetic coupling of the two Cu ions in the oxy state of these metalloproteins leads to ESR-silent behavior. Structural insight from X-ray crystallography is now available for all three enzymes, but details... 

Kimchi-Sarfaty C, Oh JM, Kim IW et al. A silent polymorphism in the MDRl gene changes substrate specificity. Science 2007 315 525-528. 

ESR-silent, suggesting low-spin Fe11. This has been confirmed by the use of Mossbauer spectroscopy. Kinetic studies suggest that a substrate-enzyme complex is formed before interaction with dioxygen. Binding of substrate has no effect upon the Mossbauer spectrum, so it is not certain that it binds to the Fe11 centre. 

E0 = -0.042 V (Pierick et al., 1993). The reduced P-cluster of Av2 transfers at least one electron to FeMocoN to form the ESR silent "super-reduced" FeMoco, (FeMoco) 1. (Ohrme-Johnson et al., 1972 Miinck et al., 1975). A combination of cyclic voltampermetry, potentiometry and ESR spectroscopy has allowed the observation in isolated FeMoco of two redox transitions with essentially different potentials, E0 = - 0.3 eV and E0 = -1.0 eV (Newton et al., 1996). The first transition of FeMoco is from oxidize state to semireduced state of FeMoco and the second transition is related to the substrate-reducing state in FeMoco during the nitrogenase reaction turnover. Independently, for the redox pair FeMocoN ( FeMoco)2", the Eo was estimated as... 

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